CA2133203C - Hybrid human/porcine factor viii - Google Patents

Abstract

A hybrid human/porcine coagulation factor VIII is produced by isolation and recombination of human and porcine factor VIII subunits, or by genetic engineering of the human and porcine factor VIII
genes. Subunits of factor VIII that have been puri-fied from human or porcine plasma are isolated, and hybrid human/porcine factor VIII is produced by mixing either porcine heavy chain subunits with human light chain subunits or by mixing human heavy chain subunits with porcine light chain subun-its, thereby producing human light chain/porcine heavy chain and human heavy chain/porcine light chain hybrid molecules.
These hybrid molecules are isolated by ion exchange chromatography.
Alternatively, recombinant DNA methods are used to swap elements of porcine factor VIII for the corresponding elements of human factor VIII to produce hybrid human/porcine fac-tor VIII.

Description

WO 93/20093 PCT/L'S93/032?5 HYBRID HQHlIN/PORCINE FACTOR VIII
The United St=ates government has rights in this invention arising from National Institutes of Health Grant No. HL 4092_ that partially funded the research leading to this invention.
Backgrouad of the Invention This invention relates generally to a hybrid human/porcine factor VIII and methods of preparation and use thereof.
Blood clotting begins when platelets adhere to the cut wall of an injured blood vessel at a lesion site. Subsequently, in a cascade of enzymatically regulated reactions, soluble fibrinogen molecules are converted by the enzyme thrombin to insoluble strands of fibrin that hold the platelets together in a thrombus. At each step in the cascade, a protein precursor is converted to a protease that cleaves the next protein precursor in the series. Cofactors are required at most of the steps. In its active form, the protein factor VIII is a cofactor that is required for the activation of factor X by the protease, activated factor IX.
Factor VIII or antihemophilic factor was noticed in plasma and named in the 1930s. In the 1940s, a deficiency in factor VIII was associated with the clotting disorder hemophilia A. Factor,VIII was found ' to be X-linked anc9 was hypothesized to be a protein.
Work involving bovine, human, and porcine plasma identified factor VIII as a protein in the 1980s, though its definitive cellular source remains uncertain.
Precisely how factor VIII functions in Moon coagulation is unknown. It is known that factor VIII
is activated to factor VIIIa proteolytically by thrombin or factor Xa. In combination with calcium and phospholipid, factor VIIIa makes factor IXa a more Vd0 93/20093 PC'ff US93/03275 ~Y~~
v . _2_ efficient activator of factor X by an unknown mechanism, People deficient in factor VIII or having antibodies against factor VIII who are not treated with factor VIII suffer uncontrolled internal bleeding .
that may cause a range of serious symptoms, from inflammatory reactions in joints to early death.
Severe hemophiliacs, who number about 10,000 in the United Mates, can be treated with infusion of factor VIII, which will restore the blood's normal clotting ability if administered with sufficient frequency and concentration. The classic definition of factor VIII, in fact, is that substance present in normal blood plasma that corrects the clotting defect in plasma derived from individuals with hemophilia A.
Several preparations of human plasma-derived factor VIII of varying degrees of purity are available commercially for the treatment of hemophilia A. These ixaclude a partially~°purified factor VIII derived from the fooled blood of many donors that is heat- and detergent-treated for-viruses but contains a significant level of antigenic proteins; a monoclonal antibody-purified factor VIII that has lower levels of antigenic a.mpuriti~s and viral contamination; and recombinant human factor,VIII, clinical trials for which are underway: Additionally, a preparation of partially-purified porcine factor VIII is available to treat patients with inhibitors to human factor VIIT, i.e., those who have circulating antibody molecules that bind and neutralize human factar VIII.
Hemophiliacs require daily replacement of factor , VIII to prevent the deforming hemophilic arthropathy that occurs after many years of recurrent hemorrhages into the joints. However, supplies of factor VIII
cancentrates have never been plentiful enough for treating hemophiliacs adequately because of problems in commercial production and therapeutic use. For i~V~ 93/20093 ~ '~ ~ ~ ~ ~ J PCT/US93/03275 _3_ example, the commonly used plasma-derived is difficult to isolate and purify, is immunogenic, and requires treatment to remove the risk of infectivity from AIDS
and hepatitis viruses. Recombinant human factor VIII
may lessen the latter two problems. Porcine factor VIII may also present an alternative, since human factor VIII is unstable at physiologic concentrations and pH, is present in blood at an extremely low concentration (0.2 ~g/ml plasma), and its specific clotting activity is low, compared with porcine factor VIII.
Since many inhibitors of human factor VIII react less strongly with porcine factor VIII, porcine factor VIII is currently used to correct factor VIII
deficiency in patients under conditions in which they do not respond to infusions of human factor JITI. A
limitation of porcine factor VIII is the development of inhibitory antibodies to it after one or more infusions, The problems associated with the commonly used, commercially available, plasma~derived factor VIII
have stimulated significant interest in the development of a better factor VIII product. There is a need for a more potent factor VIII molecule so that more units of clotting'activity can be delivered per molecule; a factor VIII molecule that is stable at a selected pH and physiologic concentration; a factor ~TIII molecule that is less apt to produce inhibitory antibodies; and a,factor VIII molecule that evades immune detection in patients who have already acquired antibodies to human factor VIII.
It is therefore an object of the present invention to provide a factor VIII that corrects hemophilia in a patient deficient in factor VIII or having inhibitors of human factor VIII.
. It is a further object of the present invention to provide methods for treatment of hemophiliacs.

W~ 93!20093 PG'TtUS93I03275 It is another object of the present invention to provide a factor VIII with an increased efficacy in factor VIII clotting assays. .
It is still another object of the present invention to provide a factor~.VIII that is stable at a , selected pH and physiologic concentration.
Mary of tire Invention A hybrid human/porcine coagulation factor VIII is produced by isolation and recombination of human and porcine factor VIII subunits or by genetic engineering of the human and porcine factor VIII genes.
Tn the preferred embodiment, subunits of factor VIII are isolated and purified from human or porcine plasma, and hybrid human/porcine factor VIII is produced either by mixture of porcine heavy chain subunits with human light chain subunits or by mixture of human heavy chain subunits with p~rcine light chain subunits, thereby producing human light chain/porcine heavy chain .and human heavy chain/porcine light chain hybrid molecules. These hybrid molecules are isolated by ion exchange chromatography.
Alternatively, recombinant DNA methods are used to swap elements of porcine factor VIII for the corresponding elements of human factpr VIII, also resulting in hybrid huanan/porcine factor VIII
molecules.
Methods for preparing highly purified hybrid human/porcine factor VIII are described having the steps of: (a) isolation of subunits of plasma-derived human factor VIII and subunits of plasma-derived porcine factor VIII, followed by reconstitution of coagulant activity by mixture of human and porcine subunits, followed by isolation of hybrid human/porcine factor VIII by ion exchange chromatography; (b) construction of domains of porcine factor VIII by recombinant DNA technology, followed by ~..y,.,. ...: , ~..:,...z .:::-; ....,:...... . .,,.,~:.. ,~~~:~:'.:.~
,~.~,'': ....,..,.- ,_. -...-;.:.~ ,-~,.~.:., ,. ~;. ."., . -_..y~, .,', ,;, y,.~..;
,~t . .. :.'. ..... , :..,:,.,. .~ -,..,; . ~;. ..:.. , , .. . :: '' .:~.:-.:-:r' ~.~' ~~% " .':...._.. _.:~ : ~. .:;:- .. ,:,.. ,; ,:. ,. .;J.. ~, , i;:, .~ ' :..,_,, :.a~ ..!>;.
1.
.r,...a......,~. ..... ... . ...... . .,.u,.~. .., .~.:,. ;" , , ,'::. .
.....".. ....... .:'.. , :......". ,..... . . ,., . ,. .. ....:

Gi4. , ' .~.::.' , ~,~.:.....,. . ,..... ,, ,.;,..,.., ... .. '.: ~ ;.:...,.
W~ 93/20093 PCT/US93/03275 _5_ swapping of domains of porcine and human factor VTII;
or (c) creation of hybrid human/porcine factor VIII by replacement of specific amino acid residues of human factor VIII with the homologous porcine factor VIII
amino acid residues by site-directed mutagenesis.
The resulting hybrid human/porcine factor VIII
has specific activity greater than human factor VIII
and equal to or slightly higher than porcine factor VIII.
brief Description of the Drawings Figure 1 (Prior Arty is a diagrammatic representation of factor VIII molecule showing the subunits (heavy and light chains) and the domains.
Detai~.ed Description of the Invention Def initions As used herein, '°hybrid human/porcine factor VIIT " denotes a functional factor VIII protein molecule with sequence derived from human and porcine factor VIII: This hybrid human/porcine factor VIII
has a specific activity equal to or greater than that of porcine factor VIII and; has activity in a human factor VIII assay. zn some embodiments, this hybrid human/porcine factor VIII is not cross-reactive with all human factor VIII antibodies.
''Specific activity," as used herein, refers to -the activity that will correct the coagulation defect of human factor VIII deficient plasma. Specific activity is measured in units of clotting activity per milligram total factor VIII protein in a standard assay in which the clotting time of human factor VIII
deficient plasma is compared to that of normal human plasma. One unit of factor VIII activity is the - activity present in one milliliter of normal human plasma. In the assay, the shorter the time for clot formation, the greater the activity of the factor VIII
being assayed.

~~ 93/20093 ~CT/US93/03275 ,..,, , ..,,4'; -l ~~~

A "hybrid factor VIII°' or "hybrid protein,'' as used herein, is a factor VIII protein in which the amino acid sequence is derived in part from human and , in part from porcine origin. This hybrid factor VIII
can be made (1) by substitution of isolated, plasma-derived porcine or human subunits (heavy or light chains) for corresponding human or porcine subunits;

(2) by substitution of human or porcine domains (A1, A2, A3, B, C1, and CZ) for corresponding porcine or human domains; (~) by substitution of parts of human or porcine domains for parts of porcine or human domains; or (4) by changing one or more amino acid residue ( s ) in human f actor VIIT to the residue ( s ) in the corresponding porcine sequence. A fusion protein is the product of a hybrid gene in which the coding sequence f or one protein is extensively altered, for example, by fusing part of it to the coding sequence for a second protein from a different gene to produce a hybrid gene that encodes the fusion protein. As .
used herein, a fusion protein is a subset of the hybrid protein described in this application, °°~aCtor VIII deficiency, °' as used herein, includes deficiency in clotting activity caused by production of a defective factor VIII, by inadequate or no production of factor VIII, or by partial or total inhibition of factor VIII by inhibitors. -I3emophilia A is a type of factar VIII. deficiency resulta.ng from a defect in an X-linked gene and the absence or deficiency of the factor VIII protein it encades.
°osubunits'° of human or porcine factor VIII, as used herein, are the heavy and light chains of the protein. The heavy chain of factor VIII contains three "domains," A1, A2, and B. The light chain of factor VIII also contains three ''domains,'' A3, C1, and C2.

W~ 93!20093 PC.T/IJS93/03275 -~~~~~~~J
- General Description of l~iethods Hybrid human/porcine factor VIII molecules that have greater activity in a standard clotting assay when compared to highly-purified human factor VIII can be constructed as follows.
Four types of hybrid human/porcine factor VIII
and the methods for preparing them are disclosed herein: those obtained (1) (a) by substituting a porcine subunit (i.e., heavy chain or light chain) for the corresponding human subunit; (b) by substituting a porcine domain (i.e., A1, A2, A3, B, C1, and C2) for the corresponding human domain; and (c) by substituting part of a porcine domain for the corresponding fragment of the human domain; and (2) by changing one or more amino acid residues) in human factor VIII to the'residue(s) in the corresponding porcine sequence. The'hybrid molecule may contain a greater percentage of human than porcine sequence or vide versa, depending on the origin of the various regions, as described in more detail below.
It is shown below that hybrid human/porcine factor VITI consisting of porcine heavy chain/human light chain and corresponding to the first type of hybrid listed above has greater specific coagulant activity in a standard clotting assay as compared to human factor VIII. The hybrid human/porcine factor VIII can be useful in treating patients with inhibitors, since these inhibitors can react less well with hybrid human/porcine factor VIII than with either human or porcine factor VIII.
Hybrid human/porcine factor VIII proteins listed above under group (2) are made by isolation of subunits of plasma-derived factor VIII, followed by reconstitution and purification. Hybrid human/porcine factor VLII proteins described under group (2) above are made by recombinant DNA methods.

_. _g_ Preparation of hybrid human/t~orcine factor VIII
molecules from isolated human and corcine factor VIII
s~bunits by reconstitution:
Hybrid human/porcine factor VIII molecules are prepared and isolated, and their procoagulant activity is characterized. One method, modified from procedures reported by Fay, P.J., et al., 265 J. Biol.
Chem. 6197 (1990); and Lollar, J.S., et al., 263 J.
Biol. Chem. 10451 (1988), involves the isolation of subunits (heavy and light chains) of human and porcine factor VIII, followed by recombination of human heavy chain and porcine light chain or by recombination of human light chair and porcine heavy chain.
Isolation of individual subunits from both species involves dissociation of the light chain/heavy chain dimes by chelation of calcium with ethylenediaminetetraacetic acid (EDTA), followed by Mono Sue" HPLC (Pharmacia-LKB, Piscataway, NJ). Hybrid human/porcine factor VIII molecules are reconstituted from isolated su,bunits in the presence of calcium.
Hybrid human light chain/porcine heavy chain or porcine light chain/human heavy chain factor VIII is isolated from unreacted heavy chains by Mono S'" HPLC
by procedures for the isolation of porcine factor VIII, as described by Lollar, J.S., et al., 71 Blood 137-143 (1988).
These methods, described in detail in the examples below, result in hybrid human light chain/porcine heavy chain molecules with greater than six times the pr~ocoagulant activity of human factor VIII.

_g_ Preparation of human/porcine factor VIII
Molecules by recombinant engineering of the sequences encoding human and porcine factor VIII subunits~
The human factor VIII gene was isolated and expressed in mammalian cells, as reported by Toole, J.J., gt al., 312 Nature 342-347 (1984) (Genetics Institute); Gitschier, J., et al., 312 Nature 326-330 (1984) (Genentec;h); wood, W.I., et al., 312 Nature 330-337 (1984) (Genentech); Vehar, G.A., et al., 312 Nature 337-342 (1984) (Genentech)), and the amino acid sequence was deduced from cDNA. U.S. Patent No.

4,965,199 to Capon et al., discloses a recombinant DNA
method for producing factor VIII in mammalian host cells and purification of human factor VIII. Factor VIII expression in CHO (Chinese hamster ovary) cells and BHKC (baby '.hamster kidney cells) has been reported.
The cDNA sequence encoding human factor VIII and predicted amino .acid sequence are shown below.

W~ 93/20093 ~C,T/US~3/03275 ~.;.., -10_ c~~
0 0 0 0 0 0 o a o a o 0 0 0 0 0 0 0 1D N GD K9' O ~ N CO ~f' O t0 N CO et' O ~.O N CO
r1 r-i N C7 f~r ~i' d' lfl 1D 10 I~ i~ ~ 01 Q~ O O
~r~-t HH,~ HHr7C7~'H~'UU C7 U~' U H ~ U H H H t,9 c7 U C.9 U ~ H ~ H
C7 H C7 U H U ~C C7 C? U C9 ~ T,~ U
~ C7 H U C7 H C7 H U C7 H U ~ U ~ U U
H ~C H H H H H C~ ~ ~ ~ H ~ E, ~ ~ H U
H(H-aU~~HCU.7~HUHH~UHU~C9 N U C7 U L7 U H U '~-a U U ~ U ~ H U
U H C7 ~ ~ ~ ~ H H H ~ ~ U U U ~ H
r0 ~ U H C.~ C9 H H U U C7 C7 C~ C9 U U
W
H ~ U C9 U 'C7 H U H U H C7 C7 C7 U
H H ~ C7 L7 H U C9 C7 C7 H H H H H
O ~C H U C9 U U ~ ~ ~ C7 U H U ~C U
U ~ U H ~ U U H ~ U U U ' M H H U t~ U ~ L7 C7 'Ca C7 U C7 H U U U C7 U ~ H C7 H ~ C7 C7 H C9 C7 ~., H ,~ H ~ U H U ~ U ~ U ~ c7 H
U H ~ U H C7 H U V t? H H ~ U t9 H C7 U
~ QaC ~ ~C U H LU7 H AC ~ U H U t9 U H ~ U
U Lh (~ H U C7 t.7 H C7 C7 t~7 U ~7 C7 U C7 H
(~ ~ . ~ ~ C7 U ~ C7 U ~ H c7 c.9 ~ H H H L7 pt ~ H H Lh H H U H U H H U C7 U U
~1 H H U U U C9 H t9 ~ U U L7 H C2 U c9 CJ1 H H ~ C7 ~ C7 ~ ~ c7 U H H ~ ~C H C7 ..,. H C7 ~ U ~ U H ~ ~ ~C L7 ~ U U U
U ~ C7 H ~ U t.~ C7 ~ C7 t9 U ~ U
O H C7 U H U H H E-~ Z7 C7 c7 H H c7 U U
U C7 ~ H U C7 U U ~ t7 ~ H C7 H U H U L7 t': ~ t~ ~ aC C7 ~ L7 C7 C? C9 C7 C7 H C~ U ~ U
U U U U U H H L~ C9 H ~ H H U H
H U H ~ H ~ H C7 H ~ ~ H ~ U c.7 ~
O U C7 C7 U ~ E-~ t9 ~ U U C9 i9 C7 H C7 U
tld C7 H ~ H H U U H E-a U H ~C H C7 C7 C7 H U (~ ~ H ~ E., U H U C7 U C7 ~.7 H
~ U L7 H ~ C7 H ,~ C9 t7 U C7 H ~ C7 ~C
~ H ~ ~ Ea H ~ U C9 H H L9 H H H E~ U ~ L7 U U U H C7 U H ~C ~ C7 ~ H C7 H ~C ~ C7 Ch -U C9 L7 U H ~ U H C7 U KC U U H H C9 L7 H t"~ C7 U E-s H H H U i.7 H ~ H C7 H C7 E-~ ~ C7 ~C H H U H U C7 ~ H H ~ H H H
H ~, U H H U H U C7 U ~G H H c9 U U C9 ~C C7 H H H H H U ~ ~ ~ ~ H U H H H ~ H ~
H U H H FC H U U ~ U C7 H U U ~ H C7 U
O U U ~, ~ E., H U U H H U ~C ~ C7 H H H
.~..~ U U ~C c7 ~ H H H ~ ~ ~ H H H C.9 ~ H
U H U U U H C9 FC t9 C9 H H U ~ H C7 U H
~ H H c_7 U ~7 U H H ~ U U H U H C7 4-t ~7 H ~ H ~ U U U U ~ U U U H U ~ C7 U
~ ~ H C7 H c9 U ~ ~ U c~ KC ~ c7 H H ~C H
H H H c7 C7 U H H H H H ~ U U H U
~ Ei H H H C7 H H U ~ ~C ~ H C7 ~ ~ H
U H U U H C~ H U U E-~ U U C7 C~ H U
U (,) r.~ U r~ ~C ~ H U ~C U U H U C7 FC U H
t9 ~ U i~ U C7 C7 U ~ C7 C7 H ~C U H t7 H H U H H ~ c7 c9 H t7 ~ H ~ U ~ ~ c7 L7 C? H H C7 c~ H ~C ~ H H Ca U c7 U
H U H E-r U E-~ H U U C~ H H H U ~ U ~C
U H H U U U C7 U C~ C~ C7 U U H ~ U H C~

W(D 93J20093 PCTlUS93/03275 ...e1 0 0 0 0 0 0 o a o 0 0 0 0 0 0 0 0 0 d~ O ~C N CO d~ O ~ N CO d' O ~O N CO d' O to r-i N N M C~ d' 1!1 t!7 lD l0 I~ CO CO 01 a1 O r-i r1 r1 e-1 e-I r-1 r--1 r! ri r-I w--I r1 r-1 r1 r-i r-1 r-I N N N
H C9 H H C7 H U H ~ ~ H U U C7 ~ U c7 U
,c~ ~ U H ~ ~ ~ U r7 H ~ C7 H C7 H U H H
U C7 ~ C7 H H U ~ H H U U ~ ~ H H C7 ~.., ,~ H C9 KC ~ U ~ U H C7 H U H H
U U H ~ ~ ~ U C7 C7 L7 ~ U U ~ ~ U C7 U
H U U U C7 U C9 C7 ~ ~ ~ U C7 H H
H H H U H (7 L7 t9 U C? U U U E.H.., ~H., E., U ,a~ H ~ C7 H H U C7 " H H C7 ~ ~C
H H c9 a C7 ~'C H c7 H U c9 C7 C9 U C9 U
c.7 U H H H ~ H U ~ ~ '~ U H ''~ '~ U ~C U
H C7 ~C H U H H ~C U H C7 H U U H U H
U ~ c7 H C7 ~ H ~ U H ~ H ~ H ~ H H H
a~ U H U H H ~C H H C7 U C7 U C7 C9 U
H ~, off, U U ~ C7 C7 ~ ~: C7 H H H ~ H
C7 L7 H U ~s U L7 H L7 U U V ,~ (H,7 H
H ~ E.., H H H U ~ t7 C7 ~ H
H H ~ U E~ C7 H U ~ H C7 U C7 H ~C U ~ U
H L7 H U ~cC ~ C7 H H C7 U C7 ~ H U U
U U E, U H H ~ H KC c7 C9 H c7 L7 e~ U ~ C7 ~ ~ H U U ~ U ~ C7 ~C H
U ~ C7 H H ~ C7 H U U C7 H U H C9 H U
U H U H ~ H t9 ~C H U U C7 H
,~ ,a~ , ~ H C7 H U U C7 ~ C7 U
U C7 C7 ~ U C.9 U ~ U U H C7 ~ t9 ~ C9 H H L7 U off' H U C7 H U H ~ ~ H H
H ~ U ~ H ~C ~ ~ H H H H C7 c9 H ~ L7 U H C7 t'? C7 CJ H U ~'C C? ~ U C7 U U C7 C7 (H,~ HC~Ut7'HC9H HH~UC7HH
C7~t~.9~U'~~H~U~t~.9~~UH
H H U t9 H C7 H H ~ ~ c7 L7 H U
~ U U c7 U H U H U c.~ ~ ~C H H C9 ~thU,L9 Lh;,~C9U~CHU~UZ7C9C7 c7 C7 H H U H ~ U H c7 c9 H H ~ H
H H H H U ~ U H KC c7 ~ ~ ~ U
U C7 U ~ C~ U U L7 C7 t9 V ~ ~ ~ Er U H H U ~ C7 H C.9 ~C H H U H .
H U U U H H U ~ U ~ ~ H H H U C7 H
U U ~ U U ~ U C7 t9 c7 ~ c7 H C7 C7 H ~ U H ~C t7 E"~ '~ U C7 ~ H H ~ ~ H U
V ~ ~ H ~ H U U U ~ H ~ ~ U H U
U U' U H U - ,cC C? U H C7 H C~ U H
H C7 C7 C7 U C7 C~ H H H ~ H ~ H H U C7 E, E.., ~ U ,~ ~ H U ~C H H U ~ ~ H
U U ,,cc~~ U U U C7 U U C7 C7 U H
U ~ ~ ~C U U U H ~ ~ U H ~ H U C7 ~C
(a U U (,7 ~ C7 U H C~ H ~ ~ H ~~aa:: H U ~ H
HE.U.,U~~UCU,7HHHCH.7~HH
r~ U U C7 H (~ ~ C~ ~C C7 L~ ~ H U U U C7 U H U H H U H ~ H U U H C7 H U ~ ~ . H ~C ~ ~ ~C c7 U U C7 H H H H
U H U L7 C7 C7 C9 E-~ ~ U ~ H H ~ H ~ L7 U H U H FC C7 U H H H ~ c7 U U U U U ~7 H U U U U C~ ~ ~ H ~ C7 ~ H H c7 H
~., H U H H H C7 U H ~ H H ~C C~ U ~ C7 H U ~ H U U C7 ~ ~ ~ ~ H U H U H
U H ~ ~ C7 ~ H U H U U ~ ~ H
~ ,~ U U U L~ H ~ H U U ~ H U U ~ U
t. ~ -..
r ~.. r , ~.x ,~):.', , ,...w, .. . .. . ... s...~...r.... . ,.,. , . . , a . ., . ..... ,.
... .. . ,.. . .. r.,... . , ..c. . .. ., . , ..... . , ..

WO 93120093 PCT/iJS93/03275 a ~~~~ -12-O ~ O O O O O O O O O O O O O O O O O
N CO d' O l4 N CO 'd' O lQ N CO et' O l~ N W c9' O
N N M d' ~' lIS tC? lD C'~ I'~ CO CO 41 O O r-I r1 N C7 N N N N N N N N N N N N N C1 f~ M c'1 l"1 M
U C7 H C7 H H L~ U C7 H L7 ~ H C7 H C7 U ~C H
H C9 C7 H U U H U H U H U H ''~ U U H U
H C7 H U ~ U H L? U U H H C7 C7 C7 ~C H
,e~ (~ H C7 U U C7 U U C7 ~ ~ H ~ H ~C H
H H C7 H C7 ~ H ~ t9 U ~ U U U U C9 H
U U ~ H ~C c7 H C.7 ~ ~ c7 U U ~ H c~ H ~ U
U H H U H H H ~ U H ~ H t9 H ~ H U H C,7 U H U ~ U U ~C ~ H C'~ H H H ~ C7 U U Z7 r.~ ~C H H U ~ L7 U H ~ ~ ~ U C7 ~ C7 ~ H
U C7 H ~ U C7 H U H ~ .a: H U ~ C7 H U
E-~ L7 H ' U ~ L7 H H U U C~ U H ~ ~ U U
U H C7 C~ U ~ ~ U ~ C7 C7 ,~ U H t9 ,~ U "~ ,~ L7 H H H c9 ~d ~ ~ H c9 ~ H
U H ~ U C7 ~ U ~ C7 H U U U U H U r1; U U
~ U H ~ C7 H C7 ~ ~ C9 H U H ' F-~ ~ C7 H H
U H C9 H ~ U H U U H H U ~ ~ H
~ C7 H H U H U U ~ ~ U H U U ~ U ~ ~ U
iht9U~H~HHL7C7~~UH H
E~-~~~~HHH~UHC.~~~UUU~
L~ U H t7 pC C7 C7 H ~ Z7 ~ U L7 H H ~ ~C ~
~ U UU U H H U H C7 H ~C H AC C7 U H,~
~'c~ UU~UU' ~HC~7tH'~~,EU-i U H U ~ ~C U C.9 H U U H H C7 H C.9 E~ U ~ U H L7 ~ H U C9 H U
C9 C7 ~ H H t9 U U C7 U U H C7 ~ H ~C C7 C7 Lh ~C7 H U ~ ~ C'J ~ H~ ~ ~H,, H U ~ H
,~CHU.~CU.7UU~H~H~ C9 U U U H ~ H KC U C7 ~ ~ ~ ~ ~ H U
U C7 ~ C7 H ~C U H ~C U U H C,7 U ~ H
H C7 C7 U H U t3 ~ ~ U C7 C7 U ~ H
t9 ~ C7 ~ ~ H ~ ~C C7 H H H H ,~ t7 ~C H C'l ~C C7 U U U U ~ C7 ~ U ~ C9 U U H
U ~ ~ C7 ~ ~C U U U U ~ ~ ~C ~ C7 H t7 ~ H
U U C) U C9 H H ~C H H U H ~ H C7 ~
(.-~ ~ ~ U U H U ~ U H U ~ U C7 U ~ ~ U
H H U ~ ~ ~ C9 U H H C7 ~ ~C H U H
U U C7 H ~ U H H H t9 ~ H H H C7 H H
E-a H t,'? ~ ~C U U U ~ c7 C9 H U ~ H C7 U H U
H C7 U H C7 L7 U U C7 ~C ~ L7 ~ L7 H ~ L7 H U
U H H H H H ~ H H U H ~ U ~ U U
~ U H C~.7 ~ U C7 U H H ~ C7 H C7 ~ C7 H c7 ~C H H H ~ f7 H ~ C7 H c9 H ~ U H U H L7 H U ~ U ~ ~ ~ ~ ~ H H
C7 U H C7 ~ H ~ H U H
H U U U U [.-.i ~ U H U U H C7 U U U H (a H H ~ U ~ H ~ U H ~ H H U C~ U H
C~ C9 U U ~ 94; C.9 U U H ~ U ~7 U C7 L~ H H
C7 C7 H ~ ~ U U U ~ ~ U Ch C~ ~ U AC C7 ~C
H ~C U U U c.~ ~ C~ C7 C7 U H C7 H ~C H ~C U U
U U ~ ~ ~ U H ~ H C~ H H ~ C7 ~C ~ C7 ~
H H ~ ~ ~C c9 E-~ t9 ~ t7 U H C7 t7 U U U t~ H U H ~ ~ ~ H H E-4 C7 H E°~ H
H H ~ ~ ~ ~ H H ~ ~ U ~ ~ ~ c.~ H H H U
H H U ~ U H U H c7 H H c9 ~ H
U ~ U U H C~ t7 U ~C E-W7 t~ U H E-~ ~ ~C
H U C7 ~ L7 C7 L7 H ~ U ~ ~ ~ ~ U H E-~
H U H L7 ~ ~ H ~ ~ E-~ C7 C7 U H H H

w~ ~3lzoo~~ ~ d ~~ius9~~o~z~s -i3-O O O O O O O O O O O O O O O O O O
1G N DO d' O 10 N CO ~T O 1,0 N N eY O t0 N OD
M e1' b' In l~ l0 i~ I~ t0 Ol Q1 O O r~ N N M M
M M M M M M M M M M M et' d' d' d' ~1' d' H ~ H C7 U ~ U ~ C7 U H ~ ~ H U U U
U H U H CH7 ~ C7 H ~ H H ~ ~ ~C H C9 c.9 "SC ~ ~C C7 U ~ H C9 H H ca U ~ H
HH~~aaU::UC.H7CH.7UULH7UC~.h ~UU~H
H KC ~C ~ ~ H ~C ~ C~ L7 U U
r~~C7~H~U~~H~~HC9~ UUU
U H H C7 ~ U H U H U H U ~ L.~ U H
U ,e~ H ~ ~ H H H H U ~ U U H
C7 t~ E-~ U H U H U H C7 ~ c9 ~C H H
H ~G C7 C7 H ~ C7 H ~ H H U H C7 C7 U U H U ~ H H ~ H H H H ~C U
H V ~ ~ ~ ~ H U U ~ H H U H H H
H ~~US.U7~~~U~H
C~.7H~~HC.~9~Ut7H~C7H~~H~C
H U H U C9 U ~ ~ H ~ U U ~ ~ U H c9 H H ~ H ~ H c7 ~ ~ c~ ~C ~C ~ ~ ~ ~ ~C
H ~ U U (7 ~ L H U U Ch C7 U H
U H U ~ U H ~ H H ~ ~ U ~ Cb ~ C7 U ~ ~ ~ U U ~ H ~ U AC C? ~ U C7 U U t9 H E' V U H C,7 ~ ~ U C7 U' C7 H H ,~C H, ;~ H C7 H ~ ;~ C.U7 ~ ~ ~C U
~.7 ~ E7 H U ~ C7 H C7 U U (~ r~ E-s C7 C? C7 ~ ~ ~ ~C U L7 H
H C7 ~ H ~ ~C ~ ~ U C7 U U U ~C U
U H C7 U C9 ~ C9 ~ C9 ~ U H C7 U
C7 ~C H H U H ~.7 H H C9 H U U U U
~7 H G ~,h ~C ~ ~ U ~ C9 H ~ H c.9 ~ H
U H t9 C~ C9 ~C H ~ H U H
~H~~U~UH~L7UHL7U(~.7~~t9 H H U ~C ~ C7 ~ H C7 C7 H ~ s.7 U ~ U C7 H ,c~ U U' U U ~ H H ~ H U U H H
iH7 ~ ~ ~ ~ C7 ~ ~ H H U ~ t~.~
H ~ U H H H H H ~ C7 ~ ~ C~ d ~ ~ U H
U U H H U U H U ~ H U H H U '~ U
AC t7 H H H H ~ C9 C7 C7 H H ~ U H H
U H L7 U U ~ H ~C ~C ~ H U C7 ~ U U C7 U H H ~ H ~ U H ~ L7 ~ U ~ C7 t7 C7 H Z7 U c.9 ~ C7 c.7 ~ ~ C7 H H ~ C9 L7 H H L7 H H H ~C U U ~ U
C7 ~C U AC ~ C7 H rC ,~ ,~ ,~ U H ~ H U
H C9 S.7 H U U d L7 ~ H C7 r.C
E~ ~ H ~ ~ ~=C ~ U H H C9 E-~
H E-U.a ~7 U ~ H E-~ H H U U ~C ~ H
C7 ~ ~ C9 U ~ U ~ ~C U H L7 ~C C7 C9 U U
U U H H ~ ~ U ~ C~ C7 ~C C7 U ~ C7 ~C U
H ~ ~C ~C C7 c.9 ~ ~ ~ C7 U ~ C.7 C~ H U t~
H C7 C7 U L7 L7 5C ~ U ~ H C9 H ~ t7 ~ H
U ~ ~ H ~ ~ ~C ~ E-a H C7 c.7 ~ C~ H U H
H [-r U U H H C~ ~ U U C7 C7 U FC H H
~C ~ ~ U ~ H C7 ~ ~ U ~ ~ C7 C7 ~ ~C U C~

W~ 93/20093 1P(:T/tJS93/03275 s. ' ~
~~s:
_i4_ O O O O O O ~ O O O O O O O O O O O O
d' O ~ N CO d' O 10 N 00 d' O 1~ N 00 d' O ~ N
e!° In tf'9 lCl LO !~ CO CO O~ 31 O r1 ~I N N f'1 d' ~f' ltl d' 'd° d' b' d' ei' d' d' gyp' d' l(1 t~ tt~ tf~ LC1 111 l!1 tf~ In U U L7 ~ U a; ~ H H ~ C7 H ~ H c7 C7 ~C C7 v.~ U C7 C7 U ~ U U C7 U H H ~ H
C7 t7 ~ ~ ~ C9 ~ H c7 C7 ~ ~ c7 H ~ c9 C9 U U H C7 U ~ ~'C H H U L7 U ~
H H L9 U ~ U ~ U H C7 H H L7 L7 H C7 c7 t9 t7 C~ U ~ C7 c7 ~C c~ H c,~
H ~ U U U ~ V H U ~ ~ ~ V C7 ~ ~ C7 H U H H C~ ~ ~ ~ E~ c~ L~.7 ~ ~ H H U ~
r1; U ~ H ~C t.7 Ei H H U U L7 C7 ~C ~ ~ C7 H U ~ U ~ dC
U C7 U ~ ~ C.9 ~ C7 C9 U ~ U V U CU7 U ~ H
H tU.7 H U U ~ H CU.7 U ~ U ~ U C9 c7 H H H
U ,c~ ~ U U U H U C7 U ~C ~ H C7 H
~ Ei U U J U FF H H ~ U C7 H H C7 U H U
C7 U U U U H H H U U C9 C7 ~ ~C ~ H C7 U
,cc H L7 C7 U H U H~ C7 H ~ H U C7 U U ~C U t9 U E~ ~ H C9 ~ H U H C7 U U H H C7 U t9 U ,~ ~.~, C7 H H H U E~ ~ U C7 ~C ~C to C7 ~ U
~ C7 H ~ H U U U U ~C t9 ~ U i7 ~ C9 U C9 Ea U U U H H C7 H U H C7 H H
H U U ~ ~ C7 H U ~ L7 H ~ ~ ~ t9 U U
U H AC C9 C7 ~C C7 U C'J H U ~ U EU-ea CU7 L~.7 ~
~ ~ EH., C~.7 U ~ (U.7 ~ ~ ~ U ~ H ~C ~C H ~ H H
H U L7 U t9 C9 U L.9 C7 H C9 C9 C7 U H
~ C9 ~C C7 U U d U H C7 ~ H U Ei U~U~C~LrH~~cH7~C~7CU7~EU-rC~-~
H Ei H U C9 H L9 H ~ ~ U ~ '~ ~ ~ U LU7 c7 U
U ~H H U E~-~ ~ U U U L7 U C7 C7 C7 ~ C.~ U
~ H U U H U ~C C7 C7 U U o~C C7 H H U H . C7 ~ U C9 H H H ~:7 ~ C7 U U U ~ ~ H ~, ~ U
,~ H (~ U ~C U ~ ~ Ei H U U Ca H ~ C7 C7 H ~ H H U H U U C9 C7 C7 U ~ H H ~C H L7 E, U H H ~ ~ U ~ H H H ~7 U H -~ C7 t,7 (9 U L7 ~ H C7 H ~ ~ U ~C H ~ ~ U
E1 ,~ U C? H H C7 U ~ H H H U H H U
U ~ U H H U U ,~ H ~ ~ ~ ~ H H H
H C7 C7 L7 ~ C7 U C7 U C~ ~C U t~ C7 H U H U
U H ~ V ~ V U U H ~ EH-ea V U. ~ U U C~7 ~ ~ ~ U H H H U C7 C7 C7 U H C7 ~ H ~ U L7 H ~ H c.~ C7 E-E-Hi H ~ ~ ~ U C7 ~ ~ U ~ C7 C.~ ~ ~ L7 ~C ~ H
E-a U U U C7 U C~ ~ U C7 C~ H U C7 U U U H
U~H~EH-~HULU7UH~~EU-~N~UUH~
H U H C7 L7 U U H L? ~ ~C C~ U U C7 ~ U H U
~C H U H ~ L7 H ca H L7 ~ H C~ H c~ U H ~
U U H C7 ~C H U ~ U ~ ~ L7 U ~ ~ U H
H H H C7 ~ H H ~ H U ~ ~ ~ ~ H C7 C~
~C H H H C9 ~ L9 H U H t~ ~ H C7 c7 U H H
H U ~ U ~C C7 H C7 U ~C H U ~ ~ c9 H ~C
C7 H U ~ ~ C7 c7 ~ ~C ~ ~ ~ C7 ~ ~ ~ C7 ~C
c7 U H C7 U H H C~ ~.C H H ~C H H c.~ H
~cC ~ C7 H ~ H ~ U ~ C7 H U U L~ H C~ ~C H
H C7 ~ C~ C~ C7 H C7 ~C U ~C U
,.. . ", -,~: , . ~. ;. ., r.;:: , :-:': ,-;: ,: :; ; . . .. . . ...:'.;; y ~ r,~-.~r .~f f, r ~,~, t ...; ,:', v ,~. ...~: ..n~,..~'. . o, ~.-~ . : ~: :. ~:, :- . ." .. . .-:, a .. ..;:, . . a ".'~ ~... ~...~;,v y~;~,.
.fly:'. ...! ~.
. . . a . ~.:l . . . , , , .
YSF....x.. . x ... ... .... . n . 7 .. . . . .. . ..... . , ... ... < .. , , ,. . . v . ~ . o .. ..... . ..

~'t:1'lLJS93103275 W~ 93120093 -i5-O O O O O O O O O O O O O O O O O O
CO Wit' O 10 N t9 ~1' O l0 N CO d' O t~ N 00 d' O
!fl l0 f~ t' GO CO C71 O O r-I ri N C'i t~'1 d' d' u1 vD
ifs u1 In tdl fIl lI1 to d0 l0 tD lD tD 10 ~D ~O l0 ~O ~9 H ~ ~ H H H U C7 ~ ~ U t9 ~ U H ~ U
H C7 (9 U C7 H ~ C7 C7 H H ~ U C7 ~ U ~ C7 H H H U U U H H c7 ~ C7 ~ ch U ~'' U U
~ ~ ~ ''~ U U ~ ~C U H C7 H C7 c.7 ~ H
~ CU7 U H U U C7 U ~ C~ U H U U
H H C7 ~C ~ C7 H AC U C7 ~ C7 H c7 H U C7 H U ~ U C7 C7 ~ H L.~ ~C " H L7 U ~ U U C7 U U C~ H ~ H c9 U ~ L.7 H L7 H H c7 L7 U C9 H U Lh U ~C ~ U ~ C7 H H H H H U
U ~ ,~ ,~ C7 L.7 ~ U L7 H U U U H C9 ~ C7 raC t.9 U U' U' ~C L9 C7 C7 U H C7 C7 C9 H U C7. H C9 H C7 H C? U U L7 H H
U H H °~ H ~ ~ ~ U ~ ~7 U U U H , ~ C7 U
H E"U., H H U C7 C.~ U C7 vd ~ C7 C7 U H U
~H~, H U a4; U U ~' H U' C9 H H C7 C7 ~C U C7 H
H E, ,~ U U ~ U ~ r.~ U C7 U C7 H C? ~ ~ H
H ~ U ~ U ~ H H E.U., E'~-a U ~ H ~ ~ ~ ~ H
H U U t9 ~ e~ U C7 aC U U U ~ ~ H C9 U ~ U H H U L9 H C7 C7 U ~C U U ~L9 C7 U U ,~ ,e~ H U ~ ~ U L9 U H H
H L9 C7 ~ C7 ~ H U e~ t9 U' U t7 H U U U H U U H U H U H H U U U
C7 H H H H C7 t7 ~ H H C9 U ~C t7 C~ H H
H ~ ~C U U H H C7 H H H t9 ~C H ~ C7 ~C H
U H H U ~ U U H t7 L7 H H C7 C7 U U U
U U ~ ,GC Cy H U ~ C7 H H H ~C ~ C7 ~ H U
U U U ~ KC U H L7 C7 ~ U U H U ~ ~C
H ~ E, ~.,, U Cj H U ~ H H H H ~ U H H ~C
U U U ~ U U H C7 ~ C7 H c.9 t3 U H H C9 s~ L7 ~ U U C9 U H c7 L7 ~ H C.7 ~-r ~ ,c~ H H U C9 ~G H ~C ~ U C7 ~ H H C,7 H
~ U H C7 U H C7 U C7 U H '~ U
H (U.7 U C7 ~ H KC C7 ~~ C7 U H ~ H ~ KC U ~C
U ~ e:7 H U H U H Ch H H ~ H H U t9 H U
U ~J H U ,~ H U ~ C~ C9 ~ ~ U H H U U
C7 C7 t7 H U H H U ~C H C~ U ~ ~ U H
C7 ,~ ~ U' U U U U ~ U U C7 U
,~ ,~ H U H H ~ tn r r v v U ~ H H U ~ H U H U C7 ~ ~ H U
H C7 C7 H U H U H H H C~ U H
C7 ~ H U c7 C7 ~ KC C7 ~ H C7 U C
H H C7 L9 U C7 U ~ U H ~ C7 C7 H U
.7 H H C7 H H L7 H U U C7 c~ U c.~U
7 ~C H U H H ~C ~ H ~ C7 H ~ U H H
r~ U H (7 U H 9 H U ~ H H C7 U H H U H
U H U C9 H ~ t~ H H H C7 H C9 H H c7 U
H ' ~ U H H r.~U U H H H H ~ H C7 ~

H c~ H H U L7 AC ~C L7 H U ~ U H H U ~ H
~ H U U C7 H c9 c7 H ~ H H H H U H
U ~ U U U ~ ~ H ~ U C~ H ~C ~ ~ U U
G ~C ~ U ~ U C7 H H ~C H C? U C~.7 H ~ ~ U H ,~ ~ H H H U H L9 H H H
~"7 (,~ U C7.C~ c.7,c~C7 ~ C7 U ~ U C7 H U
C? H U H ~ H H ~ U ~ ~ E''~ U U U
H ~ U C~ H U U H H 2 ~ ~ U H C7 ~ ~ U
H ~ H ~ ~ ~ H H ~ ~C ~ ~ ~ ~ ~ ~ H

iAr~ 93/20093 PCT/~J~93I03275 >,,~, :~~~~
6~~~
O O O O O O O O O O O O O O O O O O O
~ N t0 ~9' O ~ N CO d' O t0 N CO 'd' O l~ N t0 d' 4D r r C~0 X71 01 O O e-~ N N M C) d' In tf1 v0 i0 r mo ~ ~ r r r r r r r r r r r r r C7 ~ H U ~ C7 C7 ~C C7 U L9 H L7 U H ~ ~ H
H H U ~ H L7 U ~~ H ~C U C7 H U
H H sQ; U U U H ~ U U U U C7 H U H C9 H H H H c,7 H c~ U U U U C7 ~ U U C? rC
C~ ~ H ' H H U ~C H U ~ H H C7 U H H U
U C7 ~C U U H U H U C9 U U C7 H H ~ H
U H C7 ~ C7 H U U C7 C7 ~ ~ U U C7 ~ C7 H U ,~ U U ~ ~ U ,~ ~ ~ U U FC H H H C~
UU~H~ UUH~HL~.7C7H~HH~CU7 C7 U' U ~ H C9 ~ H U U ~ C7 ~ U ~C C7 U H
H C7 Ch C~ C7 C7 C7 C7 ~ C7 U U C9 Ca ~7 H U H
~ C~ H ~ H ~ C9 E-y~ t7 ~ ~ U ~cC H
U H ,~ ~ U '~ C7 ~ U ~ L7 U H H
U ,~ U U U U U C7 U H L7 U
H C7 ,~ C7 ~ H H U U U H U C9 U H U
U H U U U ~ H C9 H U C7 H H H
C9 C~ H H U U U U H U H C~ U ~ U U H
H U ,c~ H H H H ~ U U H ~ H C7 U L7 U C7 U
H ~ H U ~ U C9 L7 ~ C9 U H H c.9 ~ c~ ~ H
,c~ U U ~ ~ ~C C7 H t7 U ~ H U H H
U H U Lh ~C U H U U H ~ U H
U~~~UHHH~H~C7~LH7CE7~t~.9 U t7 C7 H H U H U H U C9 ~ H H L~ C9 L7 U U ~ H C~ C7 H H ~ C9 U H U U C7 U ~2 C7 H
H H H U C7 ~ U H C7 C7 C7 H C9 H ~ H U H H
UU~~UtU.7~HU' UC~7UUUHL7UHU
U H C7 U H FC U t7 ~ t9 ~ U U H ~ H H C7 U
U ~ U U U H H H U ~ H ~ t9 H
rQ; C7 C7 U ~ U H ~C U H C7 H H U U
U C7 H ~ U H ~ V ~, EU- H U U U U ~ H
H ~ ~ ~ ~ ~ H c7 U ~ U H H ~ ~ U H ~ U
H U C7 C7 L7 ~ C7 U U ~C H C7 ~ H C7 L7 E,, U U H U ~ ~ ~ U H ~ U U H U t.7 H f.7 U U ,~ H U a U U U C7 H U ~ H H U
H H C9 H U L7 C7 U U U C7 H ~C H C~ ~ U
~E-H~~U~~H~HCU7LU7U~~~~E~°~
~ C7 U U ~ U H ~ H H H C~ ~ H C.7 U H H H
U ~ U ~C ~ C9 U ~C U H H H C9 H H ~ C7 H C7 H H C7 H L9 H c7 ~ U H U ~ U H H ~C
Ea (,~ ~ r.~ acC U ~C U U ~ ~ H H ~~ H C7 ~C H ~C H ~ H U H U U U U U H H C9 H U U t9 C7 C7 H U U C7 H C7 H ~ U ~ C7 H
H ~ C7 ~ C7 ~ H H ~ ~C C7 U H H C7 C7 H ' H
U ~C H ~ U ~ H ~ U H U U H H L7 ~ H U
C7 H U U C7 U U H C7 C~ C~ U U r~ FC ~ C7 c~ U C9 U C7 H C7 H H H c9 H H ~ H H KC
C7 ~ H H U H KC H C7 C7 L9 C7 U U ~ H ~ U
H H ~ ~C U U U C~ Ca ~C H U U C~ H U
UU~(.9U~~UHHC7C7LhC7HU~i7U
H U ~ H U C7 ~ U L9 H H ~C ~ ~ U ~ H H
~C U H H U C7 H H H H U U U H ~C E--~ U
U ~ ~ U H H H U U C7 ~ C7 C~ C7 ~' ~ H
H U C7 U C7 U ~ U ~ H C7 H H U C7 U ~ U

pC~'/ ~J~93/032°7~5 WO 93!20093 _77- . ..
o ~ 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 0 O ~.O N Ca '<t' O l0 N CO 'G' O ~G N CO d' O 1D N
CO CO (31 01 O ri e-~ N N f'7 Wit' d' td) !n 1D ('~ I'~ CO
t', l'~- I'~ t~ 00 CO CA t70 CD t0 CO CO CO CO t0 00 CO
H ~C C7 U H ~ ~C C7 ~ ~C U ~ H L7 C7 L7 C7 U
r.~ H H ~ ~7 c9 c7 H c7 U ~ C7 c7 H H ~ H U
H H C7 H C7 ~ U ~C U H ~ ~ U H c7 c7 H ~ ~ H off' C7 U H U AC U H C7 FC H
(7 U U ~ C7 H U U U ~ ~ ~ U U
H H CH-r C7 U ~ L7 H H ~ U H
H t9 H H U ~ ~ ~ ~ ~ ~ H H U H
U H ~ ~ H ~C H c9 c~ H H ~ H E-i H H H
U H ~ U C7 U H H H U H ~C H U ~ U
E-a H U c7 H ~ U U U U H C7 H H Ei H E1 H C9 U H ~ ~ H U C9 ~ H ~ E-~ H
H H ~ (7 ~ ~ H U t~ C7 H U ~ C7 H H H
H U C7 H ~C H E-~ C7 U ~C C7 U C7 U H
C7 U C,7 H H H J C7 H U U H ~ ~C C9 C7 t9 H C7 H c9 ~ H U U H U C~ ~ U H H H H
~ ~ ~ ~ H U t,9 U U ~ U H C7 ~ U H ~ H
U H ~ L~ H ~ U U C9 C9 H C7 U U H U H Ch H U H H U ~ U U
C7 ,~ C~ U H ~ H ~ ~ ~C C9 ~ ~ H H U ~C
U ~ H C9 ~ U U H C9 c7 H U C7 ~ U C7 ~ H ~ U U H ~ ~ C7 U C9 C7 U ~H
H U ~ E, E., U H ~ H L9 H C7 ~ C3 ~ HH~t9C~.7 E"'HU~U(.U7~ UUH

H H ~ H U H
E-~
H U C7 C7 C7 t9 C7 C7 C9 ~ H U C7 U N H~
U U H ~C H ~ C7 ~ c7 l.~ U U C9 C~ C7 ~ H ~ N H ~ ~ v U ,~ ,~ U C9 C7 C9 H c7 H U U ~ ~ C7 C:9 H t,9 C7 U H C7 ~C U H U
U H U U H U ~ Ch U U L7 U U
H "~ U U U ~ ~ °~ U ~ U N U U ~ H
~ ~ U ~U.., ~ U H H ~ H ~ ~ ~ ~ ~ H
H H ~ C9 ~ ~ ~ ~ H ~ ~ EH-~ CH,7 CU.7 H ~ U H
H ~ U i~.7 ~ U c9 H AC ~ U H ~ H U C7 H U
~ C7 C7 C7 ~ ~ C7 L7 ~ ~ ~ U ~G C7 U H C7 ~C E"' ~ U ~ H U U H U U H ~ t~.7 L7 ~ U H C~? H H U H H ~ U U U FC
U U U H U H H H ,~ c7 c9 c~ U ~ U ~ H H
U H U H ~ ~C ~C H H U H H C~ H
U ~ ~ H ~ H ~ ~ U H U C7 U U ~ ~ ~ H
,~ U U H H H H H ~ ~ U H H H U H U
H H H H ~C H ~C C~ U U H H U ~ L~ H
U ~ H ~ ~ ~ ~ H ~ ~ U ~ ~ U H U H
H C7 U d H H ~ H ~ c~ ~ c9 H
C7 c.9 ~ H H L-~ H U ~ C7 H
U ~ c7 H C7 H U ~ U ~ ~ ~ ~ ~ U
U H H ~ EH-~ U ~ ~ E~-~ ~ ~ ~C L7 ~ U H
H U ~ ~ ~ U ~ U U ~ C7 H H H
C~ ~ ~ H H U C7 U U ~ C7 ~ C~ C.7 C7 H ,c~ H ~ ~ H E-~ ~ U H c7 FC H
U H H U U U U H U C7 C.~ U U H C

uV~ 93/20093 PCT>US93>03275 _1'g_ ,;~4~~ v x H

O O O
GO s~ O O lff O O O O O O O O O O O O O
CO Q1 O O Lea 1Q N 00 d' O ~D N CO d' O lD N CO
O"J GO 01 Cf1 r1 ri N C1 C1 d' d' lIl li710 I~ f'>

~ v ~ ~ z o' x sx a w ca H w c~ x w w w cn U z a x H c~ ~ ~c x c~ H H o a H > ~ H ~ ~x x z w w ~ w F-, , ~-r~c z x o~ a ~ a a a~ ~ z ~ a N
H c~ U ~ o a c~ ~ a w ~ w w ~ c~ r~
~, o ~ a ~ z H ~ c~ H ~n a ~ H x H w ~ c~ ~ a w z z c~ u.,cn z H W W r-1 N 'G4 v~ z ~ ?~ G~:i=. x Lw U Ot ~ ~ a x H a a ~ ~ ~ f~.,a H ~ ~ tx.~C7 C7 ~ H E-~Y~ t1',ai E--~~ U f3~
U a w cn a4 w a c~ rn H z cn u~ a U ~ ~ cn z ~ cn o cn ~ a w a cn L-~
H ~ ~ ~ ~ a 3 H a x carU ~ ~, ~ x ~ ~ H U ~ x a ~ W a ~ z x a a x w H E, ~ ~ ~, 3 x . ~ cn x a, ~ H a H
a a ~ w a 3 0~ a a U cn a n cn w O H Cn c~ ~ ~C z U 7 5' !~ ~ a ~d ~ a e-d~4 QG C7 H W
E-+E~ ~ H c9 D ~ Or W GL a H Z H ~ t~
U U ,e~ N z D x a i~ w ~ a W w cO C.7H
x E-o H ,r,~ 'C3 fsa ~ t!7 tx ~ ",~~ H t7 a a tY,H
a, x a a z a a a w H z~
~ U C.7 U w a-a~ Ot ~ ~ a U W z rd u~ v~ cry ~ a ~ A, ~ ~ cn ~ w H H U x ~ ~ a G4 ~ W H c=.,y. a w ~., ,~ E.a O GZ, C~ C-d tryH a ~ c9 ~ a ~C ~ ~
~ ~.,~-, ~ ~ x a Z ~ Ot a ~ H H ~ z ~G
P4 ua U ~C,C7 ~ ~ a $ ~ w x ~ U N w ~ a N w w a a x z x U x s w as w a w w w ~ ~ a c~ ~

H G~ Z a V7 w P.~w C~ a4 a4 U a ti) H A4 x cn x H U W a f~ a ~ H
3 rn cn H H en cA
U a ~ ~ cn x a ~ c~ w ~ a a H ~ H ~ x ~ ~ H ~n c~ H o~ ~ n a ~ ~ c~ c~ w x ~ w w a a as z U U ~ a ~ z W w ~ x x a z cn z o~
w f-~W a H ~ ~ Or H C7 a W c~
~, U U Z7 a :G ' ~ ~.''.~ H W G~ Ga, ~,"fs.~
E, ~, ~ ,-a y~ a ~a C7 W H ~C fs.ipi ,~ U7 t ~

~ W a a ~ ~ rx U U H cn w o= w c~ c~ a~ ~ x ~ c~ ~, w ti Or ~ ~ s-lH a x H a rn z a w v ~, ,~ ~ ~ a ~ a ~ x x x x w H H H
U~ H ~ ~ ~ H ~ H x a x c~ x x ~n w ~C H ~ a E-~H tz,3 x ~C H ~ ~ x C7 z U H ~ 3 W x ~G ~ tn a W C7 U ~ v~
H ~ ~, ~s cn c.~era x ~ cn u~ a x H H r~ x ~, c~ U a~ a a c~ a c~ H x ~ w a z a. cn ~ ~, ~., U w ~a c~ H x x H ~ a a cn w a U ~ c~ w a cx U or ~ x a, ~ a a ~ H ~ ~u ~c ~ ~. H x w H ~ c~ c~ of H
c~ H ~ a~ c~ ~ ~ x U a w w cn H w a ~
~ ,~ ~-, . s~ a a, x w t~ a cn x ~ a w H
~ ~ U H w ~ w a x H c~ a~ ~ a ~ w a H
~ a ~ a or u~ x c~ ~ a w o ~ v ~a v ~ r~ w a c~ c~ z x r~ ~ w ~ w x x x cn a, a a ~ ~ a a v ~ N ~ ~ ~ W C~ a a a x ca r~ a x o H x w cn ~ a x H x ~

l~~O 93120093 PCT/US93/03275 O O O O O O O O O O O O O O O O O O O
d' O l~ N CO ~f' O 1D N CO d' O 10 N CO ~f' O ~D N
Zp Q1 01 O O e-1 N N M M d' ~
r~ H r~ r-1 e-~ e-I e-I r1 '-i H r-1 r-i r-1 r-1 r-i r1 a z ~ a x c~ x x a H x H x a ~ D ~ ~ ~c f.4 O cn cn of w w z ~ cn x x cn x H H ~ x x cn a~ a ~ H H w x ~ ~ w z o w a v w ~. c.7 w c~ cn w cn or ~n t~ x H o~ w w x ~ rx x ~ or ~ ~ x w c~ x rx a a~ x ~ o cx a a H w w a z a H a a ~ w w ~., a a z ~ a z x ~ w H a ~ a w a u~ z x a w w w w ;~ H z c~ x v~ v x w cn H x ~ w' ~ x cn o ~ x x a x c~ x a ~n ~ w ~ ~ x ~ H w a ~n t7 x cn cn w cz. cn cn cn x N w x cn a N
~n t~ cn z v~ ~ a w x a w ~ ~ H ~ ~., H e~ w w H H a z a a w a cn ~
z E-l a ~ H or x a ~ ~n ~ E-~ cx x H w ~n x w ~ ~ a ~. x x H ~ ~ a c~ x a z H a ~
o H x z z w z w w H cn w c~ ~n r~ rn ~ o a H a cn z cn u~ z ~ z H o a a~ w a t~ ~~ x H
cn ~ a. w ~ a ~ x o ~ v ~ cn vi w z c~ z z H x c~ x ~~ of c~ x w x ~ a a a.~ a v ~, ~ z x z o~ a ~ cn x ~ x c~ z H ~ w o a~
cn H w z a c~ z w N w ~ a. a, a w p, cn c~ H ~ ~n o . ~ z x W a r~ cn w ~ cn ~7 fx cn x a z a cn w w ~ z a o~ GG ~ cn v A > a a ~ a z c~ va z ~ H ~ H o a o~ w z ~ x ~ a E., u7 E-~ W H ~ ~ z W H cn ~ z ~ ~;
w ~, a cn ~ z a > x o a w z A a x cx ~ w H z x w z ~ H x ~ 3 x a ~, w 3 w a ~n cn x c~ a a H a c~ x x x ~ c~ ~ d z c~ ~ e~ x z rx v H v~ x H x H cn x w x ~ x ~ ~ x ~ ~ a z ~ ~ w c~ ~n ~ v N
,a w, ~, ,a x m cn ~ H O ~ c~ ~ H ~ H O w W w H a ~ cx cn a x cn a ~ w H H c~ z w or H a ~ H w a w a. w cn E-~ x w ~ o w 3 a w z v ~ w w ~ H a z c~ w ~ o w o en x ~ ~ a H a ~ H o~ a~ a ~ ~ x - x ~ H ~ x H ,~ ~ z a x x ~ a cn x a w H H
a H x ~ a ~ w x x x H cn a x tx x ~ ~ w ~n ca x a w w x ~ N ~ a H ~n w x w ~ a a a ~ a H a, a s~ ~x z ~ c~ ~ or a a H ~ w ~ x w ,~ ~ a c~ z z x H a w cn ~ ~ ~~ x H
x w a w x c~ ~ x c~ cn w cn ~ x a, a, x H
~; z H ~ x a o N a ~ x c~ a ~ w H c~ a w H a ~ x w ~ x ~ ~ of H ~ o w z w a ~ w ~ c~ w x H H w H ~n w a w a ~ a x a a a ~ H w w a a ~n w x x x v~ x ~ 3 rx Os o~ x ca w w H z H w a c~ a, v rx a w w c~ a cn c~ cn ~n c~ x w o ~n a a~ H a H x ~ w a ~ o x w ~ x H w r~ cn c~ c~ a cx x w ~ a a ~ ~ c~ a a ~ c~ w ~ ~ H C~ H w x z x ~
w x a H a, ~ a, c~ H x ~ z c~ ~ a a a r~, ~ x z z ~ a x H ~ w cn ~ c~ ~., w w ~
a H a~ c~ a of ~ ~ x x H a H x c~ cn c~ z o~
ca ~, ~ ~n ~ ~ x ~ v~ x s~ H w z a ~ cx r~ rx cn ~ u~ w ~ o z z w w a a H ca x a. ~ x c.~
cn ~ a~ H ~n w x w x a w H w ~ ~ cn a ~ x v~ ~ t~ cn tl~ 4a W Ot ~ W tn ~C G~ ~ 3 0r tl.~ c=.~ AC
a c7 C7 ~n cn H cn H E-~ H ~ a a a Ei z of z a~
z cn a ~ z w a a x w z cn ~ w ~ w ~ x z c;~ x cn z w ~ w H x w ~ a x ~ w n w c~ a H x cn x H w z w x a a z o~ a H w c~ c~ H
x a H c~ a x a x N a~ a, z ~ a w a ~ w z s,..:
. .:. .. ,,. ,., ; ~, ; : .,. ..:': . . - v ; ': , ,..
,,....
~,:..,.. s.,. .. .... .. :, , ..,.; .: :. . ..:. . :.. .... .:. ..... . ..
,... : ,.

'~~ 93/20093 PCTf L1~93103275 6~ ~ ~ i~~~
O O O O O O

pp c,~ O tQ N CO

Q1 O r-Ir1 N N

..~ N N N N N

~ ~ ~ ~

~ H C

a ~ x o~ a x 3 a , a ~ ~ a p w r ~ x r n ~ x o w x ~ a c.
~

~

"~ a r'.(~H W U' ~ H H W

~ x , ~ U f-i:~ ~ a ~

z te z x ~

~ r a ~
n , ~ . .

~ a H ~ ~ ~

a a ~ ~ x c~ ~
., W ~ t..iO H a Od , ~ w ~ ~ ~ H

7 ~
i . W ~ ~ a ~

~ a ~ a ~ a ~ ~ N
' ~

o ~ ~ z ~ o ~ c a n ~ 3 ~ ~ ~ ~ z z x ~ ~

c7 cn ~C ~4 a a E-~

H ~ ~ D C t L~

~ ~

H C-pt W
.~

~ W c9 ~ z, ~

W
(~ ~.c, y~ H ~

Or H U' Cir Ew f~

WO 93/10093 PCf/1;S93/03275 Recombinant hybrid human/porcine factor VIII is prepared starting with human cDNA (Biogen, Inc.) encoding the factor VIII sequence corresponding to domains A1-A2-A3-C'1-C2. This cDNA lacks the entire B
domain and corresponds to residues 1-740 and 1649-2332 of single chain human factor VIII, according to the numbering system of Wood et al., 312 Nature 330-337 (1984). The B domain is deleted, since it does not appear to be necessary for biological function.
Porcine factor VIII has been isolated and purified from plasma (Fass, D.N., et ., 59 Blood 594 (1982). The amino acid sequence of the B and part of the A2 domains of porcine factor VIII, as reported by Toole, J.J., gt al., 83 Proc. Nat'1. Acad. Sci. U.S.A.
5939-5942 (1986), and the corresponding genomic DNA
sequence are shoc~m below. The coding region in the nucleotide sequence begins at position 675 (GGT CTC
TGG ...), which corresponds to amino acids (Gly-Leu-Tyr), the NH2 terminal amino acids.

'VV~ 93!20093 PCT/US93/03275 ,, H
H
H
O
U
4.1 O O O O O O O O O O O O O O O O O
Q) l4 N GO 'f' O l~ N CO d' O t0 N CO d' O ~t3 N
r-1 r-1 N M l"! d' d' !fl ~ ~ I~ t~ GO 01 ~1 O
U
O
H C7 H ~C U U H U U ~C H C7 ~ ~ ~ U U
4.a H C7 C7 U C7 H H H C7 H ~C U
H ~ H H ,~ H U ~ ~ C7 FC C7 U
C7 ~ ~ U H H ~'C C7 ~ ~ c.9 U
U U U U U H U ~ ~ H U H U U C9 H ~ ~C C7 U ~ ~C ~ C7 ~ H ~ ~ U H H U
~.-~ U H ~ U U H U U C7 H H U ~ U U
rtS eey U U ~ ~ H H H AC " c9 C7 C7 H ~ U ~: H
O ~HHHC7~~~UHC~7UHc9~cU7cU7 r~ U ~ H ~ ~ U H U U ~ C7 H E-~
N H U C7 U H H H C7 U H U H ~ H
H H ~ C~ U H C7 H H U H C7 ~ U U U
t,~~HHU~~U~H~~CH.7HH
,~ H H ~ U ~ c9 H H H H H C7 C7 C7 H
~.a oc~ U H ~ H H C7 H H ~ L7 FC H H ~ ~ U
H ~ H p C7 U H H ~ U ~ U U C7 cy~ U H ,~ e~ U C7 H U U H C7 H ~ ~ U ~ U
O ~ H U H H C~ ~C U ~ H H U U t9 C7 c7 ~ U U C7 H ~ H H U H H U ~ ~ ~ ~ C7 C-o e~ H U H U t7 t9 U H C~ U° U' ,~y E, H U U U ,c~ ~ H H U U C9 ~ U
~ ,c~ ;~ H ~7 ~ U C? H C7 ~ t7 C7 t~ H
H U H L7 U ~ C7 H L~ U ~ C7 U L7 H
rrj H H Ur ~ E'a U H H U' U U H U H U
~ U U H ~ L7 H U H FC H H C7 ~ C9 c9 U H
c0 ~ U H C7 U ~ H U C9 U H C.~ ~ ~ H U
d~1 CH.9C-Uac~.7C~7~~~~EU-~H~CU.7(H.7CU-44U~E-~ .
O ~ V F, ~ ~ H L7 H C.7 H C7 C7 H U C9 U
U L7 C7 U L7 U ~ H ~ H C7 ~ H t7 H C7 C7 .dJ ~ ~ H H H U U H U H C7 H C7 H t9 U U
U) U U ~ U H C7 H ~ C7 C7 H U ~ H ~C U H
H U H t9 H U U U H H H U H U H U C7 O 'C~ H H H ~ ~ U ~C H U H U H ~ H U ~C C9 L7 C7 C7 C7 C7 U C7 H C7 E-~ c,7 C7 H H U C9 C7 at ~ H H c~ c7 c7 H ~C C7 H H c~ r7 U U H H -U H U ~ ~ H ~C ~C C7 H C7 C7 H C7 H C7 U H ~C
~ ~ U E-r H- U H H H ~ H H U H CJ C7 U U
U H U ~ U H H C7 H U C7 ~~ ~C C7 H
t~°.NUU~ C9 U~Cc9~~'CH~Ut9~L7 N rtf H U ~C H U H C7 H t9 U U ~ C9 C7 ~ p,, H ~ p H ~ H ~ H U ~ H c.~ H 'C~ H
~ U U ~ U p r.~ ~ r.~ U C7 H H U H U
C7 H H ~ H C7 ~ U U C7 ~ c9 U ~ H C7 H
Z O ~ U H H C7 (C C7 U U U U ~ ~ H H U U
D U U H H H H ~ U L7 U U H ~ ~ U
U ~ U ~ ~ E"i ~C ~ H ~ ~C ~ U H C9 ~ C~ U
U ~ ~ C~ H H H U ~C C7 H U
.,~
~ O ~; H C7 ~ a~ C7 U H ~ C7 U U U ~t; ~ ~ H
O Z U C9 H U ~C H U H H C7 H H L~ C7 U C7 C~
H H ~.C ~ H H ~ H U H U ~ ~ H U ~ H
O ~ H U H H t~ L7 U H ~ U H U KC U ~ C7 U U C7 H C7 H H H U ~ H ~ H C7 C7 E.-mC ~ H H ~ ~ ~ ~C H ~ U L7 H H
pt H H c~ ~ U H U U H ~C C7 U H U H U H
W H ,~ U C9 U H U C9 H ~ H ~C ~ H U U
cn ~ C7 U H ~ ~ ~ H c7 ~ c~ H C? U U H ~C
C.9 ~ H U U U H U ~ C7 ~ U C7 ''~ C7 C7 ~V~ 93/20093 PCT/U~93/03~75 '' . w~~.~

U .!~

cd w N -~

U

O ~.a ~

O ~ ox ~

O e-iN N iJ1 H e-1H H

'~ L~ N ~

-,~ y ~ ~ O .i U1 ~q :. ~ r-~

~ ~ U' ~1, ~eC U
O

~

~ ~ ~ ~ U~ ~ ~ ~ ~ N U
-~ tn ui r, U N .1~ ~ C7 t!7 W ~ a C7 ,g U ~ o~

U ~ U H ~ ~ O ~ !a ~ ~ ~ rtf ?e 0 N i~ m G O
o U U H H ~ ~ ~ . " a ~ C7 ~ U ~
H
~"~

~ C7 H C9 .

H U ~ ~ ~ y~ fa ~ S..i O

C. w tT r ~ O ~ ~ ''~ ~C ~ ~ ~ 0.~ C7 a H d~ O ~ is O

H C7 U C7 . a d w ~ ~ H U7 1.a '~
u U . ~ o-~ U .., ~,, LI
E vU ~ ~ ~
i H r r ' r1 ri C: dl tn r ~ ~ U ~ O~ ~ ~ C7 C7 H
~ b t~

, U c9 H ~ ~ ~ y~ N O r~ N
~
H

H ~ ~ "'~ r-1 N
O U

~ ~ U H C7 7 o ~

~ H ~ t1 .;.~ S-r ~ 4l ~ ~ ~ o ~ ~ t ~ ~ N

.7 ~ . Q) N N .C , t N O ~r tct H ~ ~ ~ . ~N a w H c~

~ A~ U !-~ ~ N

. U , O~ ~ ~ O
t ~ U ~ U U x H AC et~ t~ c!y ~r ~ ref ~ ~

C7 C~

U ~C W
~

~' ~ N ~ ~ N ~ ~ ~

C ,. ~-a 49 , J H ~ -i O

L7 ~ P C7 W ~
.~ r U V; ~~.~ ~ ~ ~a ~ N t~, O

U ro ~ H ~

~ ~ r a t,~ ~.- ~cC
- ~ 1 ~

~O
~ ~ ~

~ t r r fd tQ o o 11 -i -I

aN ~ H c~ x >~

r~ ~ flf N (L1 tn U H U 'c3 ~ ~ H ~ r~ ~ tn a v7 tU eh U H 4 ~

U b 't3 ~ ~ .C. ?r fn ~ r d7 U C~ C7 U -.-i O a H H ~ ~ C7 a a? -ri ~

''C3 'C3 N U ~
~
~

~ ~1~ N ~, ?, al rte H
~ tf1 .a U ~ H a tI~ ~ L7 ~ ~

~ U H ~ ~ ~

U C7 U U .

H ~
~

H ~ ~
U H

C U C H > ..~ .~
.7 7 W~ 9310093 PCT/U~93/03z75 s.

;,, ~s ~s N s~ o ~ s~
o a N :~i '-i f0 ?i v ~I . .C t'ti r-i N
t0 r-~
d~

,~ ~, ,~ x a N a, c~ H x ,~ N

s~ ~a ~ o o ~ s~ ~s a. N
~n u, N v ca v ~ s~ v v ~n ~a N
~

,~ ,~ ~n x N w w a cn x ~ a ~ N

tt3 t~ N Cd i~.O v 5, ~ ?~ ~
O

-.-I cd N r-~ r-i 2f r-1 cd N .C
cr t~

x x ~cC r-W 7 > c~ x cn H
~ ~ N

~ ~r1 ~ N N ~ S~ ~ v ~ ro Wn ~

r-I N r-i r1 f~ v ,L', (U r-I v fa r1 N
O CO

['j r1 V' x x a E'-~ a H a x CJ o-a N N

LT ~ rtS N O f-1 rtf o eti r-I ~.a ca O O

~ ~ ~ f~ ~h ~I v ri S-1 fa fC1 v O
,Q, N

l.7 ~ a x a 6~, U3 ~ w x ~ ~n t'7 ~ N

N ~-1 N ?~ r--t liS O tti tf1 ~ ~~ ri ~ ?~ v -~ ~ of ca m M

a ,~ ~ ~, x a cn x ~ ~ x w ~c N

sa; ~., o N o a s~ ~ ~ o ra s~ o o r-r ~ a~ v .~ .~ v r, ro v ~ m ~ ~ Q, ,~ a E., E.., a C.7 x cn w ~ N

s~ o tr ~ ~ N rr ~ c~ ~a ~n ~n ~, ~ ~~ ~ ~ ~ ~ ~a~

cn w ~C ca ~ a ~ c~ ~ x ~ N

f1 O ~ ~ o ftf ~ ~ ~..1 ?a 21 ~ N
o o Ql N ~ v S-~I la v ri v ri ft3 N ri O W

cn ~ a a ~ x a c7 cn c7 x ~
N N

1r rn ~ r6 ~ t~ ~ v ~o ~n cn ~, ~ ~ it v .~ ~ ~ it ~ ~ rn r~ -i v~ H ~ L7 x ,~ H ~ ~ x ~ ~ N .
~ N

o., b o o it Q, o cu v so 9~ tr - .
o r~ ~--1 td N r1 ~"., .~." (~ ri f.~
1t? r~

cn ~ ~ ~ a. x ~c c~ w ~ x c~ ~ .
N

ctf ~-f tip ~-I f~ ~ N lf7 t~
It'1 ,",.~ N ,....~~ ~y ~ P-I N -.-1 rcf v ~p er c~ ~ ~ N x cn ~ ~ x N . a ~ x ' N ~ ~ ~ ~ ~ ~,,d r-I 1.1 N Y-a N
Q o . r., v ro v v ~s v -r-r v a, r~ co ~

x x ~ a x cn cn ~ W ~ tn a ~ N

o ~ ~ ~ ~ ~ ~ a ~ s~ n, s~
~ ~n ~n ~.1 r-t c(f v ~ to v N ~ v N v r1 av t~

x cn ~ x cn a c~ cn ~C =n ~, N

r~ ~ o ~ it F~ S~ o G V ~ v o o o "

r-; r-1 ~-I ~.1 !~ v .~" ~.1 N .L:. ~ 61 r, ~ .~

G1~ ~ x U7 H f3.. ~ t~ C7 cl~ N
N

o ~ ~s ~ ~a ~ n~ N N ~ ~ v ~n m r-i '-1 r-1 cC~ N N .~I ?~ r1 Q1 d~ N

p, C9 ~ ~"a C7 x ,~ ~ ~"a a C7 x N

~4~'N i~7;.;r: , . .... , . . . . , ..
.. W~ 9312003 F'C'~f/US93/03275 _z,5~ ~~.~~~3 fa O f0 ~.t fd t($ fLS PCJ Ul S-1 O~ rt1 O O
' ri N r-i f 1T3 (d f0 t~ ~ f~ ~ rt " C~ 1 ~

. x x x~ x a ..
H

~u rd b ~ ~ ~ ~a ~

'y. r~ N tLf ~(f ffS t~ r-1 .C m"~ t~ Ln O~
M v-1 , ~ ~ x x x x c~ ~ c~ x ~c ~
a M ~r ~r Ul fLS ft1 tff ~ ~ ~ ~ (a O O

N Ul '~, rti t(f cd rd ~ ~ r-~ rtS
tf1 M

,~ ~ . x x x x a~ a c~ x aM ~

s~ ~ a, ~a ro ~r ~s o ~, C; t0 N (C9 tea ((S (d S-1 N d' r-4 r-1 W

xM x x x w a~ ~ ~ a ua a~ .a~ ro ~s ~s c~ ~, ~ ~ cn a~
o o ~s a H ~ x x x x ~ c~ cn a w M ~

ftf rt3 QS N O f..~ f-1 fa td1 In rt! r-I r1 G: rd t0 at ~ ?, ~-1 N AI .G
O~ l~

~M x . x x xM x a w ~n w N ~ ~ ed c~ c~ ~ ~a O o ' (V '-l t" U7 f~S f~S f~ r1 Q) r1 N AI O
M , ~1 ~

a H ,. ~ x x ~c H cn c~ a a ~r M . ~r H

~ ~ ~~ ~ ~ ~ ref Wn Q

~ ~t r1 N f~ f0 (d :l ~-1 r-1 ri ri d' N

cn a ~ cn x x x H ~~
M ~

o, ~,, ~ o ~ ~ ~ ~ ~ o m ~a ~ 47 ro rt3 it it c~ ~ ~ ?, ~o ~ M x x x x c~ a c~
~

rti N rtf rtf rtf ~d tn ~-i uo u7 U) U7 r-i -r1 fIf fif ctf ~f 3~1 rtf ~ U) i'~ tc1 ~ ~ ,~ x xM x x x ~ ~~ ~ ~

roi p :..o t5~ ~ td ad ~d to ~ l-d ~ Q
O O

~j r-1 Li S.I Uj e0 to ftf (a r-I N r-~ S-1 QZ f'-~M . ~ ~ x xM x x c~ cn cap w ~

~ ~~ ~ ~ ~s ~ ro~

~ ~y U7 N tff rti f~ f0 N ?, (a ~ CO
N O

a aM ~ a x x x~ x c~ a ~ a~

~ a, ~ ~ it c~ ~ a, it o o G ~ r1 r f~S l~ !C$ tfj ~ r ri ~Q)e V' - N -I i . a ~ ~ x x x x fn ~ ~ sG
M t~ ~ cP' r V p 'J

~-1 (h d~$ Id fI1 ~ tn S-I ri .d-3 lid (U ~r C A) Q3 PLS (IS (a ~ M v lB N
tt~ ' a ~ . Ul x x x x a d' U7 r ..
, M ~ ~
[-l r1 ,-1 ~ (CS (Q f~ BJ ff1 O
O

t~ ~f ~j r--I (t5 (~S fCf tI3 y-I r~l l~ t!1 > a ~ x~ x x x w c~~ ~ c~

~a ~

~ o s~ ~ ~ it it ~s ca cn a~
co ox a . x xM x x ~ u~ H~ a H

W~ 93l2UQ93 P'C3'/U~93/03275 ,",.,, N ?..1 ~ ro ro ro ~ L7~ ro ro O O

~ ~ ~ a~~ ~ ~ ~ ro ~~ ~ ro ro o~ a H (J1 U' ~ ~ x L7 ~C x x l1) vD

roux ~ ~ ro ~ ~~ ro ro a~ N N ~ ~ ~ n~ ro a~ ~ ro ro ~ ~

cn ~ ~ ~ a; ~ ~ x a C~ x x ~ .o N O r-1 p, 'a., ~ ',3' i'~ ro O S-W CS ro O O

~, ~ ~a s-~ ,-~ o r, ~ ro ~ a~ ro ro a. c~

a ~, ~ ,~ c~ ~n c~ ~c x a~ cn x ~ x ~, ~

N m ~ ~ n~ t~ tr o ~ ro ~ ~.~ ro ro ~n ?ro ~ (~ UI f-d ~-a ro ~ .~.. ro ro N O OD

a a~, a ~ ~ ~c a~~ x a ~ x x~o >: ~ a1 a~ ~ N ~, ro tr it ro ro o o r-4 ri C; ~: N r-1 r1 ro ~J <"d ro ro et' N

t~ C7 . G.~ r.~ H C7 x ~ ~ x x w ~
y ~ ~ ~ ad ~ ro o ro a~ ~ ro ro u, ~

r-i r-i UI ri Q~ r1 ~-1 ro r-i V7 ro ro Lfl M

~ c~ ~ c~ a ~ w x H ~ x x ~n ~

N ~ ~a s~ >. ro ~ ~ ro ca o o ~ ro ~, ,--~ ~ a~ ~ ro rs cu ro ro r u~

~ ~ a ca cmr, cn c~ x c~ a x x ~o ~, ~ tr Wn ~ ro m ~ it ro ~n -..r o 49 ~ ro S.a .C .~ ro ?~ ro ro ro co eo ~~, a . ~ ~ wu, H x a > x~ x E, ago s~ ~ ~ ro roo ~ ro ~--~ ~ t~ .~ ~ o a~ m ro ro ro N o0 . >C r.7 cri cn x a, en a ~ x x ~, u~ ~ ~

ro ~ ~ ro ~., ro ro ~, U1 r-1 :~ ~i W --1 UI r-I ro r-I tLf ro i r~
M

c~ ~ e~ ~ ~ ~ ~ x c~ x x ~n ~

~ ~ ro ~ ~ ~y ~y N ft3 t3'~ ro ro p O

N ~ ~ ~~ ~ ~ ~ ~ roM t~ ro ro C',t (~ W LJ a x ~ x x do ~O

O U5 O ~ ro ~ ro ro tn ua .-a ~ ,~ ,.~ ~ .~ ~ ro ro ~ ro ro ~ ~r ~ ~ ~, H w~, ~ a, ~ x c~~o x x ~~ ~ ~, ~ ~ pro ro t~ ro o roo ro .~ o ,--i ?~ ~ tt5 .~ rs ~ ro S-a ro ro co ~

ca u~ c~ H cn ~ H ~ ~ x n. x ~ x ~n ~,, +mn ~ ~ o ~ ~ ~ it ro en ro ro ~

ro ~ ra ro ~ ~ r--~ ~ o~ s~ ro ~, ro ro r~

~ ~ ~n ~ c~ w ~ c~ ~ x a x x ~ ~

s~ ~ ~ N ro ro ~ o ro a~ s~ ro o o N ~ ~ .,-f r-1 '--i ~ ~! ro ri CU ro ~,~ '-i c7 cyn x ~ ~ a w x H ~n x ~

ro ~ o O N ~ o r-, ro ~ a, ro ct~ ~

~ ~ ~ ,t; -r-1 1d ~-1 tIj ro r-I S-1 ro e1' N

~ c~ a. w x ~c w ~ x r~ H x ~n w !~(3 93/20093 PC.'T/US9.~/U3275 ro ro ro ~ ~ ro ro is N ~
O O

ro ~ ~ ~ ~ v x o x ~ i~ x a c~

~ ro te ~ ~ , ~ ~ ''~ a a cron o x ,a ~ o C
r x x x~ f-, a c n H o ~a ro v ~ ~ a~

.y .~ , ro .~ ro a s r, x x x w H cn m a ~ ~o H x ~ .-ro ro ro ~ ~ cn ~ o ~ cn ro ~ ~n ro ro ro a~ ~ ~ a~ s~ cn ~, ro ~.
~ ~r a x x x cn a~ a a w ~ ago x roa ro ro s~ o ~ ~ cn oo x ~ ~ ~ ca > a ~n c7 ~

roe, ~ ~ o ~ ~a~ o ro ro ro ,~ ~, o ~ .~ ~ a~
,~ o, ~c ac x c~ w a ~ a, H a c~ ~, ~o ~ ~

ro ro ~p ro a ~ ~ ~~ ~ ~ a cam a w a, ,~ ro ro ro ~ s~ ~ ro ~ ~n o rr ro ,~, r1 r1 ri r-I Sm.l ro ro N
' x ~ ~ c c5 ~ c~ t--i ~C x ~ n co ro ~~ o ~ u~ o ~o ~ ro v~ ro a a ~

~ x x x a H c~ ~ x ~ ~o ro ~ N~ ~ ~, ~ ~ ro~

a a x ~

x ~ ~ c~ a c~ ~
~

sd ~ ro ~ ?~ a ~ w n ~

H r X ~. ~ x ~ C7 t~ v3 a C7 a >C o0 . t~

ro ~ ~ ro ~ ~ ro N ~
~

' N r- r t15 ro ~ i i x O

? X X x ~ L7 t4 ~ ~aC H ~C
C t~- co ro ro ro ro ~ ro ~ ~ ~' ~ ~
o ~ t x c~ ~ c~ c~ ~ a ~o ro ro ro ro ~ ?. ~ t~ is ~, ~ ro ir, u~

,mn H v w ~ re u~ ro ro r, ~

~ X x ~ ~ C7 a cn ~ ~7 x t~ co ro O ro ro ro ~ ro O ~ N tn O

-~ H ~S a7 -~ -~ ro ro r m x X~ ~ x x c~ ~c~ w a x x >m ra ro ro ~ .r~ ~ ~, ~ ro o ro ~n ~ u~

o ~~ ~ ro ~ ~ ro~

x ~ ~ c~ c~ ~ ~ s~ x ~o c~

W~ 93/20093 PCT/US931032?5 6~~ t1 O

~ ~ a~ c~ ~ ~ ~ f~ ~ Wr v N
r, ?~ ~ ~ o ~ r-a v W --i c7 d~ H E-~ ~ c7 c7 cn a o a~ a ~ ~ ~
;7 ~--~
0~

~a ~ ~ ~ it o o cn rn ~ tr v ~ a~ ~ a~ ~ .~ r~ ~, m x oo ~ ~n ~ a. s~ a ~ c~ ~ o ~ c~
w ~
rn ~ ~yy tlW f O ~ ~ o O ~ ~ I~ ~ t~
liS a -I r-i S..t r-1 f., f-1 r-~ U1 3d v O
x Sd x ,~ W C? 01 Oa C7 ~ ~ a cd ~"'~ Wn ~ a~ O H ~ Qa ~ LT v x ,~ ~oN, ~ a w~ "' o ~o ~ ~ c.~
~ c3~ S.~ ~ 0 1~ ~ ~ ~ v cad x ~ ~ 0 a~ H ~ ,~ a a~ a ,a ~o ~ c~ ~~
~i H
~

ro ~ ~ ~ ~a o s~ ~ ~a ro ~ rr ~ ~, v cn ~ ~ it H ~ ~ -~ w ~n ~ ~ .-a 9p ~ o ~rn ~~

~o ~ N ~, ~ ~o v ~ v rn c~~ s~
n~ ~ v -~ .~ ~ v .~ ~ ~ s~ >, x oo cra ~ H ~ c~ C4 c7 H ~ o H
a a e, ~

~ ~e v ~ f..i v td1 Sl~ v Q N , ~..yb x uy y"~ ~ ~ v r-1 UI .C. l4 ?, v O
9y ~ w a cn to ~ ~ ~, a ~n ,..., ~ O H a. O
a ~ ~ ,~f ~'f, v ~ ~-1 1d v O
~ o, ~ N ri ~.I e-1 ~ O 'tea v ..C;
'c~ '~ 'c~ H U1 r-i H Ua Qr P.~
b ~ ~ ~ ~ p~ ~cG O ~ ~ ~T
~ ~ ~ ~ r-1 N ~ C7 !n r-I ~ N
,~' t~ ~ e-1 C7 ~ fll ~ r-I C9 ~L' ~' ~ a C'7 ~', 9~ -r~i O
~ C7 r-~ O
V~ O~ t7 .'T:
e-~

p ~ ~a ~.d ~ ~-1 O y.~
fd ?~~ ~-1 e-1 O ~.1 ?~ ?~ !-d c'~ !M .L; , x a ~ ~ ~d r.~ E-~ C-~ ,~C v w E-~ _ td O ~
ae cn ~ ~

~~ ~ ~ ~ ~ ~~ s~ v s~ +~ ~~ v '.~0 00 r-! v r-! r-1 r1 N r-i .Lr" v to .C;
a co Z.7 a ~ ~ l~ ~ H H ~ O ~
c7 ~
0~ ~

Q ~ ~ ~ ~ N .u a~ o~
y"~ ~ v (n ,-.~ .~ o N ~-I r-i r-1 v O
w v cn ~ c.9 ' ~ CO r~ ~ C7 c7 va ~
O x ~ rn a ~

~ ~, Wn ~ ~ tr m a~ an v v v ~, ~ .-~ ~ ~ ~r, H
~ cn acs a c~ ~ a x ao ~
Q ~ Q fff N ZT Qr o, ~-~ ~ ~ O ?, ~-a N r~rn H l UI !
L~r L7 H r1 a ~C ~ Qr r-i ,~ ~ d en N O ~
~ ~ ~7 ~ r-1 O S~a f~ rtS S-I ~ ~ ~ N ~ ~ td ,..~ 111 Qy r1 r--I -C,' N ri r1 p,~ H ~ ~ ~ ~l U' C7 Q4 ~-! C~' ~ O
H H
~ ,"~

-.-,.,...;,;..T,--,,,..,; r,, , ~ ., ._,.. . .;r-: ~:;r- -:'::...,., . .:, ' .._ ;:_.;;,:
d,,., ~,;r.~. . a , F..
A
l, .
n..., ..a . 1.>., " ,.
xJFv.! >. aJ. I ..~ ,. ,.. . . ,....., PC1'/ US93/03275 W~ 93/2U093 r.~,i C

N

N

ri u7 ri W

ca C~

~..~

(,~

r..~

r-.1 a~

~~

-~ o ~~

n wo x iaVO 93J20093 PGT/US93/03275 sire 4 ' Both porcine and human factor VIII are isolated from plasma as a two subunit protein. ~'IG. 1 (prior art) illustrates diagrammatically the subunit structure of the molecule. The subunits, known as the heavy chain and light chain, are held together by a non-covalent bond that requires calcium or other divalent metal ions. The heavy chain of factor VIII
contains three domiains, A1, A2, and B, which are linked covalently. The light chain of factor VIII
also contains three domains designated A3, C1, and C2.
The B domain has no knawn function and can be removed from the molecule proteolytically or by recombinant DNA technology methods without significant alteration in any measurable parameter of factor VIIT. Human recombinant factor VIII has a similar structure and function to plasma-derived factor VIII, though it is nat glycosylated unless expressed in mammalian cells.
Both human and porcine activated factor VIII
(factor VIIIa) have three subunits due to cleavage of the heavy chain between'the Al and A2 domains. This structure is designated Al/A2/A3-C1-C2. Human factor VIIIa is not stable under the conditions that stabilize porcine factor V3IIa. This is because of the weaker association of the A2 subunit of human factor VTITa. Dissociation of the A2 subunit of human and porcine factor VIIIa is associated with loss of activity:
Since the nucleotide sequence of the porcine B
domain is known, full length hybrids can be constructed. Individual domains of porcine f actor VIII cDNA can be cloned and substituted for the corresponding human domain by established mutagenesis techniques. These factor VIII cDNA molecules can be cloned into expressioh vectors for ultimate expression of active hybrid human/porcine factor VITI protein molecules.

i~V~ 93/20093 P~'1US93~03275 ,....,, The complete A2 domain of porcine factor VIII, homologous to residues 3~2-740 in mature human factor VIII, was sequenced and the amino acid sequence was predicted. These sequences are shown below.
:<:. , ..: :; - , ...-. . , . , .;;v __ .,,. ~. ... , .;-..- .. . : - , _.; .
,... : : ,, ., .
"..... : . . .

1N~ 93/20093 PCT/US93/03275 -3:2-c~'~~
O O O O O O O o O O O O O O O O O
N t70 d' O ~ N CO 'd' O 10 N 00 t1' O l0 N
r1 ~-i N f") M Wit' d' t17 tD ~O t~ I~ CO Ot 01 O
i~l U U H ~ FC U U H U C9 ~ ~ ~C ~C C7 C9 U H C7 H ~ ~ H ~ H U ~C H H C,9 C? C7 H U H ~ ~ U C7 C7 U U Ca U U ~ U ~C ~ H
a~ C7 U C7 H Ch ~-+ C7 ~ H H ~ U L7 L7 U H
U U ,~ Er ~9 ~C H U U U C7 C7 C9 H
f~.n U H ~ H U H ~ C7 U U U C7 C7 H ~ U U
O aC C7 H ~ Cg U U C7 H H H H U U ~ ~C
C9 ~C C7 H ~ C7 ~ H H ~ ~ H ~ C9 H H C~
U U H H H C7 U U U U U Ca U U U C7 ~ C? ~ ~ H ~ ~ C7 ~ H H C7 H FC H H ~ C7 W
H ~ C7 U H H H ~ C~ U ~ C~ H H U ~C
V~ U C7 C~ ,4C ~ ~ U U U C9 U U U U U U
O ~ H U H U H ~ ~C H ~ ~ ~ H U C7 ~C
U H ,~ H U U H U U U H U =1 C7 ~ C7 U U H ~ E-i H U U ~ H H C7 U U C7 H
.~ ,~ ,r,~ ,c~ U C~ H r~ C7 E-; C7 H U H E-~ H U C7 ctf C9 E-f E~ U U U H U U ~ ~ H E-i U H C7 H
U U [-~ ~ U ~C C7 U H C7 H H H U C7 ~ H
~ ~UCU.7CU.h~~~~CU~~CU.7HHUU
N U ~, U U U U U C7 U U C7 H U U U H
s~ ~G C7 H H U C7 E-~ H t9 ~ ~ ~ H H U H
C7 C7 U C7 C7 E-~ U C9 C7 C9 U C7 U ~ C7 H
~c..e ,~ U U ~ ~ H U H ~ ~ U C9 U U . C7 C9 0 U H ~-n U U C~ H U U H U H E~ C7 Ei H U ~C H ~C H t9 H H H U H C7 ~ ~ N H ~ ~ H U C7 C7 U C7 U C.7 C7 U ~ U
U ~ U KC C7 C9 ~ ~ ~ C9 EH H H ~ H ~C U H
C tl~ H H U C? U C'3 U U ~ U C7 U C7 ~ U
~ ~~ U E-~ H .~ C7 C7 ~C H ~ C9 U ~ t9 C7 U H C7 ~aroE:,UUU E~L7UU' H~U~U~Ct9H
U U U '~ H U U ~ (9 C~ U
U H H ~ H U U U H C7 U
C7 ~ H U U ~C U ~ C7 ~ H ~ U C-~
(U .~.~ ~ KC C7 H C7 U C9 H U H C7 H U L7 U H
.~ O U U H r.~ U (g U H U U C7 U ~ H U U
~.-I N ~ ~C ~ H C7 H U U C~ ~C Ch U AC U
+, .8-' U C7 H ~ ~C U H C9 ~ U ~ C7 H C7 U H U , C9 H U H U U U H ~ H U
~I G~ E-~ C7 ~ U H E-~ H ~ H C9 U
U ~ Ch ~ H U KC C7 ~ ~ U C7 U U C7 C7 C7 C7 ~ O C7 U H C7 H U H ~ H c9 H H C7 C7 C7 2 U' U ~ H H oy U U ~C C7 ~ ~ H H H H E-~
rt H U ~ H C7 U ~ C7 U H C9 C7 U c~ H H H C7 ~ C7 ~C H H C7 U U U U H U H U ~ ~C H H C9 U C7 ct3 0 ~ ~C ~ L9 U ~C ~ U U c.9 H C7 ~ H C9 3.a Z C~ U H ~ U U C7 C.7 U H ~C H U C~
U C~ U H ~ C7 ~ ~ ~ Ch ~ H ~ H C~
~ p ~ U C7 ~ ~C ~ C7 C7 C7 C7 La C~ H U U aC
H U H H U ~ ~ H ~C ~ U H U H ~ U U
t110~ U H H C7 ~ C7 ~ H H H ~ C7 H H
~ W U C9 ~ U C~ C~ ~ C7 U C7 H C~ C~ ~ H
0~ cn U C7 H ~ H U ~ ~C U H U ~ ~C H H L7 ~ U C7 U FC C7 U H C7 ~ H C7 U ~ H H C9 U
U U t9 C~ H H H C7 H H H U C7 U C7 t~
U ~ H U ~C H H ~ U ~ H C7 U U H
U U ~ L7 U U C7 ~ C7 H ~ ~C H ~ ~ H
U H H ~ U ~ H U d H U U U U ~ U

g~,~gy..,,': ,. ,~...:;. ~.::r.'v:.
. 1~0 93!20D93 PC I'1US93/03275 W

a~ ~

U -' ~ N

t3' U

'd O U

~ N

ri ~

w z t$' G) A O .

O A U N O O o O O O

O r-l ~ TW D N CO 'i' O tp ' rd ~-1 H O -~ r-t rl N C1 C
f H ~ U

H W c0 'O O

~ ~ a z ~ a . n c ~a -~ ~ c n '~ ~ ~ a a a ~

a~ a r was a a w a ~ ~

~ : ~ ,~ ,~ C7 ~ E' '~

x ~n u~ ca o H

~-I ~r x ~ ~ Oi W O
S-a fU-~ t ?~ f.~ ' G~ ~ W
i~ a p ,, ~

H ~ z ~ x A

O U ~ G

A

.

H ~ O ~5 9~ (I~G~ U H
~ ~

-t ~ ~ ~ w r9'' ~
'H

U ~~ a , ~ U ~ H ~ ~ ~ ~ W

w t x ~ ~

~ a A ~4 ~

U~-~ 4 H ~ ~ a a ~, U ~ ~ v~ v~ a H

H a~ ro u~ ~ a a~ z c~ H

~ '~~' ~ ~ ~ A ~ ~

~ ~ c.
~

~ ~ .. ~ ~ ~ ~'a ~

~ ~
~

~ ~ ~ ~ w ~ -U ,~ ~ : ~

E., tJ rf .~ ~ ~ ~ H a ~ D
x U U~ Ca a 3 H ~ cn ~
~

- ~ ~ ~ z W x p b ar H ~ ~
o U H ~ UI D W C4 a4 4~: x U
~

....

U a C7 ~ ~ t W
~ ~ -t tn O '~'! U) C7 C7 ~
N

x ~' H z a a~

x 3 ~

tJ~ O H ~: L~

~ ~ ~ ~ I

N :4 ~, ( II
G~

d ~ t4 ~ .1-~ W 6~ ~-1~ C7 ~

x x w H ~ w H H -~ ~ z ~

,~~ ~n U x ~ ~ x ~ ~ H

~ ~ cu x o ~c~~ ca ~ x w H U ~ n~ H ~ ~ x U ~ ca z ~JVfJ 93/20093 PCT/LJS93f03275 s n~~~c~ _34_ a - phaxma~ceutical Compositions Pharmaceutical compositions containing hybrid human/porcine factor VIII, alone or in combination with appropriate pharmaceutical stabilization compounds, delivery vehicles, and/or carrier vehicles, are prepared according to known methods, as described in Remington's Phaz-maceutacal Sciences by E.W. P~iartin, the teachings of which are incorporated herein.
In one preferred embodiment, the preferred carriers or delivery vehicles for intravenous infusion are physiological saline or phosphate buffered saline.
In another preferred embodiment, suitable stabilization compounds, deli~rery vehicles, and w carrier vehicles include but are not limited to other human or porcine proteins such as albumin.
Phosphola:pid vesicles or liposomal suspensions are also preferred as pharmaceutically acceptable carriers or delivery vehicles. These can be prepared according to methods knawn to those skil2ed in the,art and can, contain, for example, phosphatidylserine/-phosphatidylcholine or other compositions of phospholipids or detergents that together impart a negative charge to the surface, since factor VIII
binds to negatively charged phospholipid membranes.
Liposomes may be prepared by dissolving appropriate lipids) (such as stearoyl phosphat~.dyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline; and cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container. An aqueous solution of the hybrid human/porcine factor VIII is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.

WO 93/20093 PCT/US93l0327S
=35 The hybrid human/porcine factor VIII can be combined with other suitable stabilization compounds, delivery vehicles, and/or carrier vehicles, including vitamin K dependent clotting factors, tissue factor, and von Willebrar~d factor (vWf} or a fragment of vWf that contains the factor VIII binding site, and polysaccharides such as sucrose.
Hybrid human/p~rcine factor VIII can also be delivered by gene therapy in the same way that human factor VIII can be delivered, using delivery means such as retroviral vectors. This method consists of incorporation of factor VIII eDNA into human cells that are transplanted directly into a factor VIII
deficient patient or that are placed in an implantable device, permeab~.e to the factor VIII molecules but impermeable to°cells, that is then transplanted. The preferred method will be retroviral-mediated gene transfer: In this method, an exogenous gene (e.g., a factor VIII cDNA) is cloned into the genome of a modified retrovirus. The gene is inserted into the genome of the host cell by viral machinery where it will be expressed by the cell. The retroviral vector is modified so that it will not produce virus, preventing viral infection'of the host. The general principles for this type of therapy are known to thaw skilled in the art and have been reviewed in the literature (e.g., Kohn, D.B., and P.W. Kantoff, 29 Transfusion 812-820, 1989).
Hybrid human/porcine factor VIIT can be stored bound to vWf to increase the half-life and shelf-life of the hybrid molecule. Additionally, lyophilization of factor VIII can improve the yields of active molecules in the presence of vWf. Current methods for storage of human and porcine factor VIII used by commercial suppliers can be employed for storage of hybrid human/porcine factor VIII. These methods include: (1) lyophilization of factor VIII in a __ ~......_~., . -~. .:,...-~..--~.--~. . , , ;~.z ,T;., . . ." :-., f .. > ~.
<f--: .

!~V~ 93/20093 ~CT/US93/03275 ,~,G.

~s:~ _ 3 6 _ .~;~~
partially-purified state (as a factor VIII
''concentrate" that is infused without further purification ) ; ( 2 ) immunoaf f inity-~purif ication of .
factor VIII by the Zimmerman method and lyophilization in the presence of albumin, which stabilizes the factor VIII; (3) lyophilization of recombinant factor VIII in the presence of albumin.
,Additionally, hybrid human/porcine factor VIII
has been indefinitely stable at 4° C in 0.6 M NaCl, 20 mM MES, and SmM CaCl2 at pH 6.0 and also can be stored frozen in these buffers and thawed with minimal loss of activity.
- ~geth~d of Treatanent Hybrid human/porcine factor VIII is used to treat uncontrolled bleeding due to factor VIII deficiency (e.g., intraarticular, intracranial, or gastrointestinal hemorrhage) in hemophiliacs with and without inhibitory antibodies and in patients with acquired factor VIII deficiency due to the development of inhibitory antibodies. ~'he active materials are preferably administered intravenously.
F,dditionaliy, hybrid human/porcine factor VIII
cain-be administered by transplant of cells genetically engineered to produce the hybrid or by implantation of.
a device containing such cells, as described above.
In a preferred embodiment, pharmaceutical compositions of hybrid human/porcine factor VIII alone or in combination with stabilizers, delivery vehicles, and/or carriers are infused into patients intravenously according to the same procedure that is used for infusion of human or porcine factor VIII.
The treatment dosages of hybrid human/porcine factor VIII composition that must be administered to a patient in need of such treatment will vary depending on the severity of the factor VIII deficiency.
Generally, dosage level is adjusted in frequency, duration, and units in keeping with the severity and P~f/US93/03275 w~, 9VV~ 93/20093 s.,:.:,.>.

duration of each patient's bleeding episode.
Accordingly, the hybrid human/porcine factor VIII is included in the pharmaceutically acceptable carrier, delivery vehicle, or stabilizer in an amount sufficient to deliver to a patient a therapeutically effective amount of the hybrid to stop bleeding, as measured by standard clotting assays.
Usually, the desired plasma factor VIII level to be achieved in the patient through administration of the hybrid human/porcine factor VIII is in the range of 30-100 0 of normal . In a preferred mode of administration of the hybrid human/porcine factor VIII, the composition is given intravenously at a preferred dosage in the range from about 20 to 50 units/kg body weight; the interval frequency is in the range from about 8 to 24 hours (in severely affected hemophiliacs); and the duration of treatment in days is in the range from l to 10 days or until the bleeding episode is resolved. See, e.g., Roberts, , H.R.; and M.R. Jones, "Hemophilia and Related Conditions - Congenital Deficiencies of Prothrombin (Factor II, Factor V, and Factors VII to XII)," Ch.
153, 1453-1474, 1460, in HematoloaY, Williams, W. J., et al., ed., 199 0. Patients with inhibitors may .
g~q~aire more hybrid human/porcine factor VITI, or patients may require less hybrid human/porcine factor VIII because of its higher specifis activity than human factor VIII: As in treatment ~rith human or porcine factor VIII, the amount of factor VIII infused is defined by the one-stage factor VIII coagulation assay and, in selected instances, in vivo recovery is determined by measuring the factor VIII in the patient's plasma after infusion. It is to be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the _ .._. .. ,. .:.~. . .. ___; .. ,; ,_,;; ; t ... ,,: , .. ::. ~..: .:,. ......
-. .; ..

WO 93/20093 P(.'1'/US93/03275 a:
c v ~~a~.:~ _ 3 8 _ administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
Treatment can take the form of a single intravenous administration of the composition or periodic or continuous administration over an extended period of time, as required. Alternatively, hybrid human/porcine factor VITI can be administered subcutaneously or orally with liposomes in one or several doses at varying intervals of time.
The hybrid human/porcine factor VIII molecule and the methods for isolation, characterization, making, and using it generally described above will be further understood with reference to the following nan-limiting examples.
example l: Assay of porcine factor VIII and hybrid human/porcine factor VIII
Porcine factor VIIT has more coagulant activity than human factor VIII, based on specific activity of the molecule. These results are shown in Table II in Example 4. This conclusion is based on the use of appropriate standard cur~res that allow human and porcine factor VIIT to be fairly compared. .
Coac~ula~ion assays are based on the ability of factor VIII to shorten the clotting time of plasma derived f~rom a patient with hemophilia A. Two types of assays were employed: the one-stage and the two-stage assay.
In the one-stage assay, 0.1 ml hemophilia A
plasma (George King Biomedical, Inc.) was incubated with 0:1 ml activated partial thromboplastin reagent (APTT) (Organon Teknika) and 0.01 ml sample or standard, consisting of diluted, citrated normal human plasma, for 5 min at 37°C in a water bath. Incubation was followed by addition of 0.1 ml 20 mM CaCl2, and the time f or development of a fibrin clot was determined by visual inspection.

...~~0 93/20093 P(.'T/tJS93/fl3275 A unit of factor VIII is defined as the amount present in 1 ml of citrated normal human plasma. With human plasma as the standard, porcine and human factor VIII activity were compared directly. Dilutions of the plasma standard or purified proteins were made into 0.15 M NaCl, 0.02 M HEPES, pH 7.4. The standard curve was constructed based on 3 or 4 dilutions of plasma, the highest dilution being 1/50, and on logo clotting time plotted against logo plasma concentration, which results in a linear plot. The units of factor VIII in an unknown sample were determined by interpolation from the standard curve.
The one-stage assay relies on endogenous activation of factor VIII by activators formed in the hemophilia A plasma, whereas the two-stage assay measures the procoagulant activity of preactivated factor. VIII. In the two-stage assay, samples containing factor VIII that had been reacted with thrombin were added to a mixture of activated partial thromboplastin and human hemophilia A plasma that had been preincubated for 5 min at 37°C. The resulting clotting times were then converted to units/ml, based on the same human Standard curve described above. The relative activity in the two-stage assay was higher than ~.n the one-stage assay because the factor VIII
had been preactivated.
Example 2: Characterization of the functional difference between human and porcine factor VIII.
The isolation of porcine and human plasma-derived factor VIII and human recombinant factor VIII have been described in the literature. Fulcher, C. A., and T. S. Zimmerman, 79 Proc. Nat'1. Acad. Sci. U.S.A.
1648-1652 (1982); Toole, J.J., et al., 312 Nature 342-34? -(1984) (Genetics Institute); Gitschier, J., et al., 312 Nature 326-330 (1984) (Genentech); Wood, W.I:, et al., 312 Nature 330-337 (1984) (Genentech);

WO 93/20093 PCI'/US93/03275 t " ~a~~

Vehar, G.A., et al., 312 Nature 337-342 (1984) (Genentech); Fass,~D.N., et al., 59 Blood 594 (1982);
Toole, J.J., et al., 83 Proc. Nat'1. Acad. Sci. U.S.A.
5939-5942 (1986). This can be accomplished in several ways. All these preparations are similar in subunit composition, although this is the first description of the functional difference between human and porcine factor VIII, not noted previously in part due to the lack of use of a common standard by which to compare them.
Far comparison of human recombinant and porcine factor VIII, preparations of highly-purified human recombinant factor VIII (Cutter Laboratories, Berkeley, CA) and parcine factor VIII (immunapurified as described in Fass, D.N., et al., 59 Blood 594 (1982)) were subjected to high-pressure liquid chromatography (HPLC) over a Mono Q"' (Pharmacia-LKB, Piscataway, NJ) avian-exchange column (Pharmacia, T~nc : ) . the purposes ~f the Mono Q'°° HPLC step were elimination of minor impurities and exchange of human and porcine factor VIIT into a common buffer for comparative purposes: Vials containing 1000-2000 units of factor VIII were reconstituted with 5 m1 HZO.
Hepes (2 M at pH 7.4) was then added to a final concentration of 0.02 M. Factor VIII was applied to a Mono Q'"' HR 5j5 column equilibrated in 0.15 M NaCl, 0.02 M Hepes, 5 mM CaCl2, at pH 7.4 (Buffer A plus 0.15 M NaCI); washed with 10 ml Buffer A + 0.15 M NaCl; and eluted with a 20 m1 linear gradient, 0.15 M to 0.90 M
NaCI in Buffer A at a flow rate of 1 mljmin.
For comparison of human factor VIII (derived from plasma and purified by Mono Q"' HPLC) and porcine factor VIII, immunoaffinity-purified, plasma-derived poxcine factor VIII was diluted 1:4 with 0.04 M Hepes, S mP~i CaCl2, 0.01% Tween-80, at pH 7.4, and subjected to Mono Q~'~''' HPLC under the dame conditions described in the previous paragraph for human factor VIII. These .. dV0 93/20093 PCT/US93/03275 procedures for the isolation of human and porcine factor VIII are standard for those skilled in the art.
Column fractions were assayed for factor VIII
activity by a one-stage coagulation assay. The average results of the assays, expressed in units of activity per AZgo of material, are given in Table I, and indicate that porcine factor VIII has at least six times greater activity than human factor VIII when the one-stage assay is used.
TAeL~ s: co~tpARasorr of xANO ~eoROxxE
FACTOR ~III COAGtTLANT ACTIVITY
Activity (U/A28o) Porcine " 21,300 Human plasma-derived 3,600 Human recombinant 2,400 Example 3: comparison of the stabilaty of human and porcine factor VIIIa The results of the one-stage assay f or factor VTII reflect activation of factor VIII to factor VIIIa in the sample and possibly loss of formed factor VIIIa activity. A direct c~amparison of the stability of human and porcine factor VIII was made. Samples from Mono QT" HPLC were diluted to the same concentration and buffer composition and reacted with thrombin. At various times, amples were removed for two-stage coagulation assay. Typically, peak activity (at 2 min) was l0-fold greeter for porcine than human factor VIIIa, and the activities of both porcine and human factor VIIIa subsequently decreased, with human factor VIIIa activity decreasing more rapidly.
Generally, attempts to isolate stable human factor VIIIa are not successful even when conditions that produce stable porcine factor VIIIa are used. To demonstrate this, Mono Q~'~" HPLC-purified human factor VIII was activated with thrombin and subjected to Mono S'~" cation-exchange (Pharmacia, Inc.) HPLC under conditions that produce stable porcine factor VIIIa (Lollar, J.S., and Parker, C.G., 28 Biochemistry 666, 1989.
Human factor VIII, 43 ~g/ml (0.2 ~M) in 0.2 M
NaCl, 0.01 M Hepes, 2.5 mM CaCli, at pH 7.4, in 10 ml total volume, was reacted with thrombin (0.036 ~M) for 10 min, at which time FPR-CHiCl D-phenyl-prolyl-arginyl-chloromethyl ketone was added to a concentration of 0.2 ~M for irreversible inactivation of thrombin. The mixture then was diluted 1:1 with 40 mM 2-(N-morpholino)ethane sulfonic acid (MES), 5 mM
CaCl2, at pH 6.0, and loaded at 2 ml/min onto a Mono S' HR 5/5 HPLC column equilibrated in 5 mM MES, 5 mM
CaCl2, at pH 6.0 (Buffer B) plus 0.1 M NaCl. Factor VIIIa was eluted without column washing with a 20 ml gradient from 0.1 M NaCl to 0.9 M NaCl in Buffer B at 1 ml/min.
The fraction with coagulant activity in the two-stage assay eluted as a single peak under these conditions. The specific activity of the peak fraction was approximately 7,500 U/A~. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the Mono S~' factor VIIIa peak, followed by silver staining of the protein, revealed two bands corresponding to a heterodimeric (A3-C1-C2/Al) derivative of factor VIII. Although the A2 fragment was not identified by silver staining under these conditions because of its low concentration, it was identified as a trace constituent by 'uI-labeling.
In contrast to the results with human factor VIII, porcine :Factor VIIIa isolated by Mono Sue' HPLC
under the same conditions had a specific activity 1.6 x 106 U/A~$o. Analysis of porcine factor VIIIa by SDS-PAGE revealed 3 fragments corresponding to Al, A2, and A3-C1-C2 subi.inits, demonstrating that porcine factor VIIIa possesses three subunits.

= -43-The results of Mono S'" HPLC of human thrombin-activated factor VIII preparations at pH 6.0 indicate that human factor VIIIa is labile under conditions that yield stable porcine factor VIIIa. However, although trace amounts of A2 fragment were identified in the peak fraction, determination of whether the coagulant activity resulted from small amounts of heterotrimeric. factor VIIIa or from heterodimeric factor VIIIa that has a low specific activity was not possible from this method alone.
A way to isolate human factor VIIIa before it loses its A2 subunit is desirable to resolve this question. To this end, isolation was accomplished in a procedure that involves reduction of the pH of the Mono S~' buffers to pH 5. Mono Q~'-purified human factor VIII (0.5 mg) was diluted with Hi0 to give a final composition of 0.25 mg/ml (1 ~cM) factor VIII in 0.25 M NaCl, 0.01 M Hepes, 2.5 mM CaClZ, 0.005% Tween-SOM at pH 7.4 (total volume 7.0 ml). Thrombin was added to a final concentration of 0.072 ~M and allowed to react for 3. min. Thrombin was then inactivated with FPR-CHzCl (0.2 ACM). The mixture then was diluted 1:1 with 40 mM sodium acetate, 5 mM CaC h, 0.01% Tween-80, at pH 5.0, and loaded at 2 ml/min onto a Mono S' HR 5/5 HPLC column equilibrated in 0.01 M sodium acetate, 5 mM CaClz, 0.01% Tween-80, at pH 5.0, plus 0.1 M NaCl. Factor VIIIa was eluted without column washing with a 20 ml gradient from 0.1 M NaCl to 1.0 M
NaCl in the same buffer at 1 ml/min. This resulted in recovery of coagulant activity in a peak that contained detectable amounts of the A2 fragment as shown by SDS-t~AGE and silver staining. The specific activity of the peak fraction was ten-fold greater than that recavered at pH 6.0 (75,000 U/Azso vs. 7,500 U/Alsa). However, in contrast to porcine factor VIIIa isolated at pH 6.0, which is indefinitely stable at WO 93/20U93 P(.'T/LJS93/032'75 '~1~. '~~~~ I ~ 4 4 ~~'~~ v 4°C, human factor VIIIa activity decreased steadily over a period of several hours after elution from Mono S''". Additionally, the specific activity of factor VIIIa purified at pH 5.0 and assayed immediately is only 5% that of porcine factor VIIIa, indicating that substantial dissociation occurred prior to assay.
These results demonstrate that both human and porcine factor VIIIa are composed of three subunits (A1, A2, and A3-C1-C2). Dissociation of the A2 subunit is responsible for the loss of activity of both human and porcine factor VITIa under certain conditions , such-as physiological ionic strength, pH, and concentration. The relative stability of porcine factor VIIIa under certain conditions is because of stronger association of the A2 subunit.
Example 4: Preparation of hybrid human/porcine.factor VIII.
Porcine factor VIII lzght chains and factor VIII
heavy chains were isolated as follows. A 0.5 M
solution of EDTA at pH 7:4 was added to Mono Q'r°''- , purified porcine factor VIII to a final concentration of 0.05 M and was allowed to stand at room temperature for 18-24 h: An equal volume of 10 mM histidine-Cl, mM EDTA, 0:020 v/V TWeen 80, at pH 6.0 (Buffer B), was'added, and the solution was applied at 1 ml/min to a Mono f"' HR 5/5 column previously,equilibrated in Buffer A p~.us 0.25 y~I NaCl: Factor ~1III heavy chains did not bind the resin, as judged by ADS-PAGE. Factor VIII light chain was eluted with a linear, 20 ml, 0.1-0.7 M NaC1 gradient in Buffer A at 1 ml/min and was homogeneous by SDS-PAGE. Factor VILI heavy chains , were isolated by mono f"' HPLC in the following way.
Factor VIII heavy chains do not adsorb to mono f'y' during the purification of factor VILI light chains.
The fall-through material that contained factor VIII
heavy chains was adjusted to pH 7.2 by addition of 0.5 M Hepes buffer, pH 7.4, and applied to a mono QT"' HR5/5 WO 93f20093 PCTf~S93/03275 ..,,, HPLC column equilibrated in 0.1 M NaCl, 0.02 M Hepes, 0.01% Tween-80, pH 7.4. The column was washed with 10 mL of this buffer, and factor VIII heavy chains were eluted with a 20 mL 0.1-1.0 M NaCI gradient in this buffer. Human light chains and heavy chains were isolated in the same manner.
Human and porcine light and heavy chains were reconstituted according to the following steps. Ten dal human or porcine factor VIII light chain, 100 ~,g/ml, was mixed in 1 M NaCl, 0.02 M Hepes, 5 mM CaCl2, 0.01% Tween-80, pH 7.4, with (1) 25 ~C1 heterologous heavy chain, 60 ~,g/ml, in the same buffer; (2) 10 ~C1 0.02 M Hepes, 0.01% Tween-80, "pH 7.4; (3) 5 ~Cl 0.6 M
CaCl2, for 14 hr at room temperature. The mixture was diluted 1/4 with 0.02 M MES, 0.01% Tween-80, 5 mM
CaCl2, pH 6, and applied to Mono S'"' Hr5/5 equilibrated in 0.l M NaCI, 0.02 M MES, 0.01% Tween-80, 5mM CaClz, pH 6Ø A 20 ml gradient was run from 0.1 - 1.0 M
NaCI in the same buffer at 1 ml/min, and 0.5 ml fracti~ns were collected: Absorbance was read at 280 nm of fractions, and fractions were assayed with absorbance for factor VIII activity by the one-stage clotta.ng assay. Heavy chains were present in excess, because free light chain (not associated with heavy chain) also binds Mono S~"; excess heavy chains ensure that free light chains are not part of_the -preparati.cn. Reconstitution experiments followed by Mono S''" HPLC purification were performed with all four possible combinations of chains: human light chain/human heavy chain, human light chain/porcine heavy chain, porcine light chain/porcine heavy chain, porcine light chain/human heavy chain. Table II
shows that human light chain/porcine heavy chain factor VITT has activity comparable to native porcine factor VIII (Table T), indicating that structural elements in the porcine heavy chain are responsible '.9'p~~'7 ...v.ewu a, ';'. ': '. ; .n ... : '~.'.' ,..,..,.... :..." .. ....... ..;.;. , ~. ':, . ~l~y-;.., .... ~:~:~. °'.:, .. ,;'. ".. .'. "' ' " .
F .. .. . . .. .... ...' ....: . ,..... . . .. , , ..r . . . ' . . .,. ., . . _. . _ ,, .. . .: . . . , ., . , .; . ..:; . . .; , , . ,., ,. .. .. . ,.. . . .~
WO 93/20093 PC'T/US93l03275 ~2 :~~~~~~ -46-t~ i~' ~
," .
for the increased coagulant activity of porcine factor VIII compared to human factor VIII.
~~a~~ II: co~tp~Rlsort o~ ~t~RID ~U/~oRC~N~
Fl~c~oR ~ggl ooA~uAc~I~z~~
NI'd°H HONA3~ AID PORCINE FACTOR VIII
Activity (U/Aa8o3 Porcine light chain/porcine heavy chain 30,600 Human light chain/porcine heavy chain 44,100 Porcine light chain/human heavy chain 1,100 Human light chain/human heavy chain 1,000 Example 5: Isolation and sequenca.ng of the A2 domain of porcine factor VIII.
Only the B domain and part of the A2 domain of porcine factor VIII have been sequenced previously (Toc~le, J.J., et al., 83 Proc. Nat'1. Acad. Sci.
LT.B.A. 5939-592 (1986}). The genomic DNA secquence of the porcine factor VIII B domain and the cDNA sequence f~r the entire porcine factor VIII A2 domain are disclosed herein.
The porcine factor VIII A2 domain was cloned by reverse transcription of porcine spleen total RNA and PCR amplification; degenerate primers based on the known human factor VIII cDNA sequence and an exact porcine primer based on a part of the porcine factor VIII sequence were used. A 1 kb PCR product was isolated and amplified by insertion into a Bluescript"
(Stratagene) phagemid vector.
The porcine A2 domain was completely sequenced by dideoxy sequencing. The sequence is as described above.
Example 6: Preparation of recombinant hybrid human/porcine factor VIII
The sequence of human factor VIII has been described in the literature (Toole, J.J., et al., 312 Nature 342-347 (1984) (Genetics Institute); Gitschier, -4?-J., et al., 312 Nature 326-330 (1984} (Genentech);
Wood, W.T., et al., 312 Nature 330-33? (1984) (Genentech}; Vehar, G.A., et al., 312 Nature 337-342 (1984) (Genentech}). The sequence is as described above.
Making recombinant hybrid human/porcine factor VIIT requires that a region of human factor VIII cDNA
(Biogen Corp.) be removed and the homologous porcine cDNA sequence inserted. Subsequently, the hybrid cDNA
is expressed in an appropriate expression system. In these experiments, for example, the entire cDNA
sequence corresponding to the A2 domain of human factor VIII is removed by oligx~nucleotide-mediated mutagenesis, a method commonly known to those skilled in the art (see, e.g., Sambrook, J., E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, Chapter 15, Cold Spring Harbor Press, Cold Spring Harbor, 1989).
The steps wire as follows: E. coli CJ236 cells were transformed with Bluescript'~' phage containing the human factor VITI oDNA insert. Single-stranded Bluesc~ipt''"/human factor VIII circular DNA was produced with M13Kt77 ~nelper phage and then purified by standard methods (Sambrook, J., E.F. Fritsch, and T.
Maniatis, Molecular Cloning: A Laboratory Manual, Chagter 4, Cold Spring Harbor Press, Cold Spring Harbor, 1989). A mutagenic oligonucleotide was synthesized corresponding to the 3' end of the A1 domain and the 5° end of the A3 domain:

5' CCTTCCTTTATCCAAATACGTAGATCAAGAGGAAATTGAC 3'.
Additionally this oligonucleotide provides a SnaB1 restriction site that can be used to insert the porcine A2 domain. On hybridization of this oligonucleotide to single strand BluescriptT"'/human factor VIII, the region between the A1 and A3 domains, i.e, the A2 domain, was "looped out." The resulting heteroduplex was extended to circular, double-stranded WO 93/20093 PCT/U~93/03275 ,..;, DNA by use of T7 polymerase, ligated, and used to transform E. coli XLl--blue"' (Stratagene) cells.
Transformants were' screened by isolation of phagemid .
DNA from several colonies, Xhol digestion' and examination of the size of phagemid DNA by agarose gel .
electrophoresis. Three clones were identified that were shorter than human factor VIII/Bluescript''" by 1 kb, as expected for deletion of the 1 kb A2 domain.
The results were confirmed by sequencing across the boundaries of the Al and A3 domains.
The porcine A2 domain has been inserted between the A1 and A3 domains of the human factor VIII cDNA by ( 1 ) PCR amplification of the ~5orcine A2 domain; ( 2 ) gel purification of the PCR product (agarose gel electrophoresis of the PCR product producing a band visualized by ethidium bromide staining, followed by excision of the band and purification of the DNA to remove agarose and other contaminants); and (3) ligation .by using T4 DNA ligase of the porcine A2 cDNA
to the human A2-domainless cDNA linearized by using, the SnaB1 restriction site. The primers used for PCR
amplification of the porcine A2 were as follows:
5' primer: 5' GTAGCGTTGCCAAGAAGCACCCTAAGACG 3' 3' primer: 5' GAAGAGTAGTACGAGTTATTTCTCTGG
GTTCAATGAC 3'. ..
The 3' primer contains nucleotides corresponding to residues 736-740 of the porcine factor VIII protein sequence,(at the C-terminus of the A2 domain), and residues 1649-1656 of the human factor VIII sequence (at the N-terminus of the A3 domain). The A3 sequence residues were included because the looping out procedure removed these residues. The ligated product Haas used to transform XLl-Blue cells, producing several colonies that contained the desired porcine A2 insert when analyzed by PCR. The product contains an unwanted thymine at the A1-A2 junction as a result of the PCR amplification of the porcine A2 domain. This °4~"
single base can be looped out by use of the mutagenic oligonucleotide 5~ CCTTTATCCAAATACGTAGCGTTTGCCAAGAAG 3' , and the product can be cloned exactly as described above (Under Example 6, paragraph 3) for the production of human A2-deficient cDNA.
Cloning of the porcine A1, A3, C1, and C2 domains is feasible with the same strategy that was used for cloning the porcine Az domain. Fragments of these domains can be cloned by the looping out mutagenesis technique. Excision of the corresponding domains in human factor VIII and any fragments thereof, including single amino acid eliminations, is feasible by looping out mutagenesis as described above. All possible domain replacements, fragments of domain replacements, or single amino acid residue replacements are possible by this approach.
The biological activity of recombinant hybrid human/porcine factor VIII can be evaluated initially by use of a COS-cell mammalian transient expression system. Hybrid human/porcine cDNA can be transfected into COS cells, and supernatants can be analyzed for factor VIIT actwity by use of one-stage and two-stage caagulation assays as described above in Example 1.
,Additionally, factor VIII activity can be measured b~
use of a chromogenic substrate assay, which is more sensitive and allows ,analysis of larger numbers of samples. This essay has been described (Lollar, P., G.J. Knutson, and D.N. Fass, 24 Biochemistry 8056-8064, 1985). Similar assays. are standard in the assay of factor VIII activity (Wood, W.I., et al., 312 Nature 330-337, 2984; Toole, J.J., et al., 312 Nature 342-347, 1984). Expression of recombinant factor VIII
in COS cells is a standard procedure (Toole, J.J., et al., 312 Nature 342-347, 1984; Pittman, D.D., and R.J.
Kaufman, 85 Proc. Nat'l. Acad. Sci. USA 2429-2433, 1988). The human factor VIII cDNA used as starting WO 93/20093 PC ~'/U~93/03275 l i ~~~~ - ~ ~ -V
material for the recombinant molecules described herein has been expressed in COS cells yielding a product with biological activity. This material will be used a standard to compare hybrid human/porcine factor VIII molecules. The activity in the assays is converted to a specific activity for proper comparison of the hybrid molecules. For this, a measurement of the mass of factor VIII produced by the cells is necessary and can be done by immunoassay with purified human and/or porcine factor VIII as standards.
Immunoassays for factor VIII are routine for those skilled in the art (See, e.g., Lollar, P., et al., 71 Blood 137-143, 1988).
Sequences of human and porcine factor VIII likely to be involved as epitopes (i.e., as recognition sites for inhibitory antibodies) can be determined through use of commercially available predictive computer programs; such as MacVector (IBI Corp., New Haven, CT). Sequences of porcine factor VIII that are not antigenic comgared to corresponding (homologous) antigenic human sequences will be identified, and substitutions-will be made to insert porcine sequences and delete human sequences according to standard recombinant DNA methods. zt is already known that porcine factor VIII reacts less than human factor VIII
With some inhibitory antibodies; this provides a basis for current therapy for patients with inhibitors.
After the recombinant hybrids are made; they will be tested in vitro for reactivity with the Bethesda inhibitor assay. Those constructs that are less reactive than native human factor VIII and native porcine factor VIII will be candidates for replacement therapy.

W~ g312009~ Pt.'T/~593/U3~75 _5~_ H
H

N

,~ .-O

e-1 (~J N

(r., O 3a U

UJ O O

O O

~

r-~I ~, ~) ~ ~

~ ~ ~ ~ fn b O N
~

-ri (a A
~ H

A ~ 'C3 ~ ~ b S3: 1 ~

Cx ~ ~ ""~ ~ L ~
O ~
?

f -..i .t . W W
~~ .. y.a .C , ~ U

. , H AC ~
, ~ ~ O ~

O ~ ~ ~ ~ ~ U ~ -13 ~ I~ H W 1 re cd W 6~.,~ ~ O~~W ~ ~ ~~~

~;.. ~ p4 r-I W cd ta., Gir z z ch E.9 N

z W A .i rtt .-mt1~ t~ .~ O ~ O z , 1",~, O U D~,,C?.i,~d' WW?at~ H H ~, ..
w cazw ,~ ~ H z ~c o ~ ~~ a ~wH~nw H
~z ,. ,--t~ H W :. ..~ ftf !~ far ~ O L-~ H O H
O ~ ~ ~

z ~-i~ . O W W :-c ~-t ~ ~ = C7 U a-i H H ~
z ~ o .. ~ U

O O ~ W Ot U W .N C9 ' ~ E~ PG H H G1 ~C H 4 ~ z W H

H ~ 6x~ W z ~n ~ ~ o ~C W H tx ~ ~ tx, U ~
, . In W En H t~ W ~ ~ H f~ U H U ~7 ~,:, H M ~ p H a H z H z cra o ~1 W W W H L~4 ~ ~ W~a ~e~H
~ ~~H~
E-i w zdY.W~Eiz' Ht~ l '' Z (s, O O CI P; E-i r. W f=. W.a ~ Gtr ~ ~-AC D 0.~ G1: L1 ,' ~C

O ~ O . ~ la H H H W W O W H d~ ~
O H O H a t= U a: tn ~ cn U el~ H ~ U O W ~ w '~ ~
U N tla U

.~ .~ ~-. .,~ ~ .~.. ,~ p: .-. ~
H ~ .~ .-. .-. O .-. ,..
~..

p H L~ ~ CO U L ~ ~ f~l p4 ~ H ~ t~
W tz. U C1 t~ U

a ., H ~ p ~,' w.r v o v 'r v.,n~wr v (% ~ ....
~ v r v ..,r ~c H z v v v ~' w ~. ., .~ ~. ....

W ~. . .~ -~ -.-c ~ -.---~ ~ .,~... v v n W~ 93/20093 PCT/US93/03275 s sa~l~~

U U

U H

ecy'C7 H c.7 U H

U

U

t7 U

U

H

U U

~ H

U U

E., ~ ~

c E
U 7 -~

~ ~ U

E
-~

C~ U

~ C~ H

r~ ~ U
a ~ W z U

C
N e ,7 m e. z ~ ~

e. a ('~ W ~ ~. H

a ~ -1 Ot U E~

ee~... l~[fl : .e fa O (l r W
-i r6 ~

P~ ~ 9 r 1.n ~ f 1fl ~ ~ ~ ~ ~ ri H ~ '~ S"~.. ri QI O
~ ' U

~ ~ H ha E- N U ~ ~ ~: O z ~ C7 +a ea S.r ~ ,..I
u1 ~-1 W ~ O c!~ c6 b I t1) t~
'a Q ~ z ~4H z h-t :C~ ~ IU U G~ o H
s1 N
o0 f~ U O ~ U .. ~ ~ .~.1~ H U U

z , z ~ z G7 W o -~1 L1 I O H
d cn ~
er H .~.a p D O H Ei :'~ N U ~ z C4 W W U C7 o tn r-1 itj H 'w H U w1 r-1 z W W H C7 En W -1 ~ H
' e!' H W ~ ~ Y'~ U z o Q a ~ w U
W o 0 s z W a ~ W ' tx ~
Ea U ~C W ~ O ~ a z t~ Z

~ Uoac v~ ~ ~wc~ w w acs wa~~iz O E~ W H x x t~ 0 ~ ~ ~ o H W C7 U
~ W
r~

~ ~ !~: z ~, L4' U Ea z r7 E-~U W En cJ~ D Ch U
W z a aW H W a W O C9 W O H ~ ~ ~ H U
W ~ Ei a C7 W 7 U

~ ~ C7 Gia ~ ~-.lGt, W z ~ W H z z e- U , C7 H7 W l W W AC A4 U
W ~ H O H

W W W W W z U ~- x cn w z o z z z ~ ~ O H z ~ H cn D H
H E-~

C4 U p W U H 1 ~ H

~ ~ U
W H a ~ .~ .-~ W O H t9 C9 ~
.~ ~ .-.
~

H~c~U ~~m H o~~xU~ ~..aw H ~ H~c~ a ~ U
~ v W
~

~..~ w v ~e W W v a v Q ~ ~r vs v ar ,~ H cn ~ x ~ s~ o O r;, ~ ...,.~ ~. U C7 > ri -~ U C~
~ x -~

-, -i ~ .. -.-~-.-i~ ~ X ~ U
.I . .-~~ v H ar . y.r v U

,- v ~

.... E-~U

- .,W~ 93>20093 PC°~'/US93/03275 _~3_ ~~.~p~~Da 0 o a o 0 0 0 0 0 0 0 o a o 0 0 0 00 -c1' 0 1D N CO d' O t0 N O~ d' O ~O N UJ ("1 r1 N M f"1 d' d' tI? 1~ ~ t'~ i~ CO 0~ 81 O O r~t r1 <-I r~
H ~ ~ ~ U U ~ H U C7 ~ ~C ~C ~ C7 H
H L7 H ~ H H U ~ H H c7 c7 U
,e~ ~ U C~ C9 U U C7 U U ~ U ~ ~ H
C9 H C9 H t9 ~ H H ~ U C9 c7 U H
C7 ~ H L7 ~ H U U U C7 C7 C9 H
H U H ~ C7 U U U C9 C7 U U U ~ C7 LH.7 H ~ tU.7 ~ Ea EU°~ ~ H ~ C'H.7 H H ~ c7 H H H C7 U U U U U C7 U H ~ C7 ~ H ~ ~ C7 ~ H H C7 H ~ H
CU7 ~ ~ ~ H H ''C c:9 U ~ C~ H H U ~C c9 ~C

U H U H ~ H ~ ~ ~ ~ H U C.7 ~ t7 L7 ~ H U U H U U U H U H C.9 H H U U H H C~ U U L7 H L9 C7 ~ U L7 H ~ C7 H C7 H U H H H C7 C7 H U
H U U U H U U ~ ~ H H C7 H C~ H U ~7 H ~ U ~ c~ U H c7 H H H U c~ ~C H c7 H
C7 tU'~ ~ ~ U U U c7 U H H C7 H ~.7 ~ U ~ ~ C7 H H U U ~ H
U U (,~ U C7 U U C7 H U U U H U H
H H U c.9 H H C7 ~ ~ ~ H H U H AC H U
(~ U C7 C9 H U L~ C7 C? U C7 U ~ c9 H H U
(~ U ~ ~C H U H ~ ~ U C7 U U L7 C9 U U
H () U C9 H U U H U H H C~ H c.9 U

H U (9 L7 U t7 U C~ U U ~ U c~
C9 ~7 ~ ~C', vc~ U' H H H ~ H AC U H ~ U
U U U C~ U U ~ U C9 U L7 ~ U U
H a~ C7 C7 ~ H ~; C9 U ~ C9 C7 U H C7 H
U U ~ H C7 U C7 H ~ U ~C U ~C E9 H H U
H C7 U C7 ~ H U U ~ C7 C7 U ~ E"~
H H AC H C~ C7 C7 H c9 c.9 ~ ~C ~ H
H U U ~ U ~ L9 ~ H ~C U H ~
U H ~ U U H U H L7 H U C~ U H C7 H ~ U C7 U H U U L7 U ~ H U U ~ C9 ,~C H U H U U ~ C7 ~ t~ U ~C ~ U '~
~,, ,r,~ ,~ U H C7 U U H U H ~ [.U-~ C~ ~ U ~ C~.7 H ~ U ~ U
ecZ U H H H
E-r U o~C C9 ~ KC U ~ U U C9 C7 C9 C7 ~ H L7 H U E., U H !~ H C7 H H C9 C7 L.9 H H
U ~ U ~ V E-H~ ~ H H ~ ~cC H
U E.., E., H t7 ~ ~ C7 ~C H H ~ ~ C7 U U
C7 U H U H U ~ ~C H H C7 ~~aa;; U U
,~ tJ U ~C ~C U U L~ H C7 ~ H C7 H ~ U U L.~ t~ U H ~C H U C7 ~ U
C7 ~ ~ AC C9 ~ H ~ H C7 ~ C7 C7 'C,9 C7 C7 L7 t9 H U U ~ ~ C7 ~C
H H U ~ ~ H ~ ~ U H U H ~ U U H U
H U ~ C9 ~ H H H ~C C7 H H ~ ''~ U
U C7 t.7 C7 U C9 H t7 C~ ~ ~ H H
H f'w H U ~ E' U H U ~ ~ H H
~ CU7 U EH-~ C7 ~ H C7 U ~C H H C7 U H C7 i~ C~ H H H Ca H ~ H H U t9 U L7 c7 H H
,c~ ~ H U ~ H H U ~ H Z7 U U H ~ H
U ~ C7 U U C7 !~C L7 H ~ ~ H ~C ~ H C? ~C
(~ ~ H U ~ H U U U U ~ U H U

P~'/U~93/03~75 nr~<<., _54_ .; a ~ b' ~' ~ ~

O
a ~ ~ ~ ~ a ~o ~~ ~ ~ ~ ~

, s~ s~ a~ ~ o '- U7 (n H
c'1 ~ O tT

r~

H W ~' ;~ C7 t1 ~,, ~ r-1 U1 t13 . O

(-.r V.~ C~

~ Q ~ ~ ~

r1 ,<t ?a .t".ar-1 lI1 x ~ ~ [~ H t~

r-1 ~I '~ S.1 'J~a v-t N ~ ~ C9 H
~

' z u H ~C try N

A ~

b ~ ~ ~ ~ ~

C
H G~ 7 .. V ~ ~ ~

~ ~ ~ f ~ ~ ~ ~

a a . -~ N .G' ?, . r ltl ' ;~ ~ ~ ~ a ~ a w ~ ; ~, :~

, . .~ a z ~
~, ~ .~ .~ ~
cn ~ r c,v~ ~ ~

o ~ ~ ,~
~

as o.. ~ ~ ~ ~ ~ ~1 LH1. W ~ ~ ~ ~ aa.~w ~
U ~

~ ~ r -~

M; 0 U x~ ~ a ~C
~ H

w ~ W ~ w ~:~H
~
~

as c o ~ ~
n bw ~ w H ~z~ o ~ ~'o ~ a~
~~

a HN ~n a zwdw w H z H a~~ w w a w w z ~ AG U N ~ ? ~ ?
H

U w a~ , ., ~., , H o ~ :~ cn w ~oH ~

o ~a~v,~ ~ H w a ~ aM a a ~ H , H ,,'~ ~-. ~ ~
.:. .-, t .~

N W ~ G4 o ~ ~ ~ ~' d ~ ~ ~ ?r U D ~ '~ o ''' "' ' " cn ~ t7 C-~ E-r ~

~ ~ x ~ w o ~ ~
' =.-:~ :~ ~ ? ' x ~. U cn a Ei ~
,.~

~ ~ .

H v .~ v ,...

~. : ; , -~ ..:., . ..;. - -:. ": ;;: a .. ...; ~,;: ..,, ., >r ..x .<
..:-s;~.,.
,~. , ":.. r ..,. " ,. ,., . ..... ... . .., ., ,... . . . , .._ , ." ... .

!~~ 93!20093 PCT/~JS93/03275 _55_ ~~~a rss .a.~ ~ t~ ~, ~ ~ ~ v o o v eo ~ ,-, ~ ~ ~n ~ ro w ~r FC ~ ~ ~ H C9 C7 L7 ~ a-i ~ c,7 ~ N

.d ~"~ ~l ~ m ~ N ~
~ N

r ~ l t' .1 ~ ~ ~ ~ H H H ~ ~ ~ ~ tt~
.~ N

~ ~o m ~ ~'' ~ ~ ~ v ~ ~ ~o ~ , a a ~ a c~ w a ~n a c~ N

<n oa ~ ~ s~ ~, ~ P, v ~a ~ ,--, ~n ~

'~y ~ ~ .Ca .Ca r! f-1 ftS .~ e-~ r! fa N O '' a r.~ a H H c7 ~C D Oa KC C7 .
~ N ~-~ ~, u~ sa ~ s~ ~ f~ t~ ~ ~ s~
a o G ~ -~ a~ v v ~ v ~a ~ v v ~ N

, . x H~ a v~ ~ ~n ~N ~ a w H

~y N t17 Tll N f~ ~a v I".. Q1 tf1 tsl ~ ~ .~y yy '~ r~ Ul .Lb ~ ~t,' N v 131 ~''~

H H , e-a U ~L' ~C C4 C7 t?r cA a f-l ~ N

:3 r-I ~ v :3 fly v O O

r..i o rd f.1 ',~ ~.a v rti v r-'1 r-1 ~Jl r-I
f~ tri t-~ oo C7 E~ H ~C a a a ~ C7 ~
~ N

a ~ ~ ~ O tar fd v G O N ~a t!1 ~ tPl >, 3.a t~ v ~-1 to ~i .C ?~ ~
co a x c~ a ~, ~ cn H ~C G~ w H N
~ .-i (/I ~? ~ Ua :W -i ~ ~?r r'~ ~.a O O

'Yy ,t', tl~ 'fir e-1 flf r1 N tl~ ~-!
N O ' a pa , pa ~S', a (9 'J' C7 ~' ~~" H
a N
,-~

~-1 S-t ~ ~.I 5..1 ~ UI fb fa ~ ~-I 5-1 i!1 !C9 (; 'fir N .C". ~ ri ?~ U~ r~ ri raw ~'t M r1 " H a H ~ tn C7 a ~ ~ N C7 H H
H

p, ~ :3 ~ t!! :~ f.d ~ ~ l7 w'~s ~S
O O

~ ~ ~ r! a~ v '?v ~.1 v f.! r-1 r1 tIl (~l ~, a c~ a a H ~ a w ~a ~
~ N

~~ ~ ~

~~ ~ ~ ~ ,~ , C7 op ~ ~C C7 H ~C U a H C7 ~
~ N

r-r boo ~, o o v vo ~ ~ ~ v ~o ~s ~, ~ ~ s~ ~ ~ ~n ~ v o ~o ~ H c~ w w H H ~ H a H c~ N
~ ,~

~ o o ~ ~. t~ ~ s~ s~ ~, ~ rn u, ~n t~ v ~ v v v v ~ v ~-s rn w a c~ c~ a ~n v~ c~ ~n x ~ ~

~ ~, ~ v o sa ~ ~ .N ~ o ~a, cn -, ~, ..~ ~ r, cn v v v ~-1 u~ .~ v ,-~

c~ ~ x ~a ~c cn a ~ c~ ~ x a .~ N

~ a~ ~ o ~ ~ o .~ ~ o .a~ ~n 1n u!

v v s~ r..~ v ~ v m ~ v a, ~- N

H cn a cz, C~ cn G~ ~ ~C w ~ U
~ N

dV~~ 93/20093 PC1'/US93~03275 ~n~
,~ S

N ~

a H x H

r1 r~~ '~ lfS ri M

x o U aM c U~ ~ 7~ N 3~ a a a H~ w a ~ ~

a~~ ~ ~ ~ ~, ~~

' a H

w N cn .. ~ 'c~
w .a M

~ ~

,~ .~ ~ ~
o H wM ~ a ~ H

~ ~
M

~ ~ ~ ~

U t1, a H H ~C
M

, V~ 1~ ~ ~. ~ a M

1-! ~ w C~.e 9r S.1 O

o.
~

~
7...f ~ ~ ~ ~ ~ ~ .. U 'd LT ~
~ b ~J
C; ~ V! ~I r"~ ~ M H ~ 'i ~' . a IQa /~ (~ (/~ o. E1 (~
N ~

o a rtf fs r-I U7 r-1 41 p: U G :~
r-t . t,~ ~ H a G1 w ~ :~ cra to o p H M .~a H E~ o U tJJ U O z x w ~i ~ ~ ~ ~ Qr' ~ U o r-1 z W R~
lfI W

UJ tU b-1 tl1 N O~ Q1 U z O U .. y N

~w~ ~ a z w a~H
~

..

~ ~ ~ ~ ~a U x x ~ O ~ ~ ~ O Ha w U ~ U U ~ .~ ~ U Ea .. H U w H tl~ z p ~ z a a C7 cn a ~ a, O c~ W ~ O a-i ~ aC cn M

c~ wzw w w H Z H ac~cn a~~ ~ ~ a, a. ~~ Uw~NO a w w z ~x~, a~, a~ ~n ~n ~u, z za~~~ ~ x cn w zoo WN (~ ~ ~ ~ UM p w W p ~

l C7 L7-H ~ .~ r... I
..~ .-..

~, ,~~ ~ ~, ~, o H oz,~mUO a w H ~ ~~cw N ~ ~ ~ '~ ~ w ar Wr ~ ~ ~ v v ~ o..ws ~ w c~ U a. ~ ~n ~ x ~ w o N

-+ ~ ~ a~ o ~, ~ ~

~ ~ ~o u~ v ,-, w - - -~..~ '~', J M ,Q', ~ H Z " '"i'~ "'~"' f...~ .....,.~.,.. ....

N

f.,." i. ~ :::' ~ , .:::.un >:~ . . , :. .. .... ,. ': ' . . ... '::.
,vf ,:....... ,...., .,..,...t: ,...:... : ,........ .........r~~.
~......~..,.. ,.:v.., .....:... .,.:.. .,...~"..:.
......'...............,.,..;~.:.. .....:-.. .........,: ~........: ,.,.,.. .."
:.
:vt.n..:.,. ..'.. ~ ,..:....,....... r::-r": v.,; ... ..- ,.,.......~.
......... . .:...,.:~... ,...,.. ..::.r ~' -.~: . . ..<... ... .
.............,... ::.."....,. . .,.,.: ........v:: . ...,. ...... ,..
.......:.. '.
l _4,n,. . ., .. .... . " ,.... . ,... . . ... .. . .
~~t. t 1 . t,. .. .. . " t .... . ..., . .r... . ~, < .. .. . . , ... ,... ...
n. ... , W~ 93/20093 P!C'11'llUS9310327~
-S7- ~~~~~~J

t0 N 00 eY O 1C' N CO eP' O ~D
ri ~-I N C'i C: d' V' to tD ~D
~~EH',~UHUUC7HU
UE''~U,~HCU7~HUH
H U H~ C9 U ~ ~ ~ H H H
' y0",i 10., ~ ~ U H ~ C7 C? H H U U
.~ ~ H ~ U C7 U t9 H U H U
~ U CU7 U iHJ ~ U U U
[.U-~ ~ H ~ c~.7 ~ U~ H ~ .
~-~ ~ H~UE~-~rH7E~-~U~H~U
-,,~.,I ~ .~ .~ ~ c9 t9 U H C7 C9 U U H H
rcJ ~ tC c0 ~ ~ ~ aC U H C9 H ~ ~ c7 ~ N p p .. (7 C7 L7 H U C7 ~ C7 H C9 C9 _~j~ ~'~ M~HHC~.'?C-U°~HUHU
N Q ~, ~.., U C7 U U H C7 II U 11 ~ Z H H ~ ~ ~7 ~C C7 ~ ~ d, U
OI' OI H L7~CU~UH
a.s r'r La ~C U ~ C7 H ~ C7 U
~ ~~ H ~ H c7 U H U H H H C7 >'"i t'", La F'.'s t9 oct, H U t,'3 U U d', C7 a' ~ cn W ~ W O~ ~ C7 ~ ~ t9 ~ C7 C9 C7 U
E-~ U H U H ~ H ~ H
,~,~ ~ O O w t~ O O .. ~ ~ C7 ~ UH ~ ~ H H U
H ..1,! y N H '1-~ ~ r I~v-i~~ UNH 0 H U ~ L7 ~ H ~H'3 CU7 ~ ~ +'' H U c7 H RC C9 H
>~ H H ~C H ~ U ~ ~ ~ CH..h ~
~ tn d5 ~ r-t t~ ~ W U C7 ~ H
~ H o r",~ H [~ r,,~ U H U H U" U AC, U
', ~ Z 9 ' U H O U U ~ H H H H U L7 ~ o H .,.~ W o H -ri G~ ~ C-i ~' C7 ~' H H U H U C7 x H ~ ~ H ~ W U H H U H U r7 U ~ H
'~ H 04 t3' 'e H ~ pT' f~ U t9 U L7 H U C7 H C7 U
W ,~ W W W ,eC W W H H H H H U ~C ~ C7 FC H
~~Ux WHUHH~HUU~CUC7 W ~ O H W ~ O H U U U H ~ H H U U H H
~ ~ as 0 ~ ? ~ 0 ~ U U ~ C~ ~C H H H FC
W H U U U H U ~ U U H
H .,.. ..~ ... H ~. ~. ....
~ ~ t~1 L'~ ~ ~ til O Oi CH.7 EH-a ~ H ~ U U U U ~ U
W v .,.,r v W °-~ ~ '~.i ~' ~~HHHtU.7CU7UHHH
H H H H C7 H H. U ~ ~C
C7 H C7 U H t~ H U U H U
.,~.i -x x i.7 ~ U C7 U C7 ~ C7 U ~ ~7 ~", ~ -.r ~ H ~ H H U H H U U
U H H U U U L7 U C~ C7 L7 WO 93/20093 P~1'/US93/03275 r 0 0 o a o 0 0 0 0 0 0 0 0 0 0 0 0 0 °
N a> er o mo N co ~r o vo N co ~r a ~ N eo ~ o Cp Q1 Cf1 0 o r1 N N M C'1 d' ~~1 ll~ ~7 t-d r-i r-1 s-1 r9 r1 r1 ~ e-1 ri r1 r~1 e-1 r1 U U ~ U U ~ H t7 H H C7 H U H ~ ~C H U
~.7 U ~ H H H ~C ~C U H ~ ~ ~C U C7 H ~ c7 C7 ~ L7 U ~ U C9 ~ C7 H H U ~C H H U
~ Ca ~ C9 U H KC H C9 FC ~ U FC U
U ~ U L7 H U L7 U H ~ ~C ~ U C7 C7 C9 ~ U
H U H ~ ~ H U H U U U t7 U C~ C9 H ,~ U H U ~ c7 H H H U ~ H C7 c7 ~ C9 U C9 U ~ U ~ H U H C9 KC ~H ~ C~ H H U C7 H H
U U U ~ E-i H H L? ~ C7 ~ H L7 H U
La L7 C7 U U U U H H H ~ H U
Ei U C7 C7 U H H C7 ~ H U H H ~ U E-~ C7 H H H H H U ~ C7 H C~ ~ H U H H
U H U ~C U u~ U H U H H ~ . ~ H H
C7 U C7 ~' ~ E-t ~° ~ U U ~ ti L7 ~C ~ C7 C7 Ca C7 C7 U C7 U C7 C7 H L7 H U C7 ~ H t7 U
C7 C7 H c~ ~7 H ~ H H H H U ~ t7 C7 ~ H
t7 H H H H H ~ U H L7 H U ~ H C~ U
U ~ to H E-a C7 H U AC C7 H H L7 U
HU~CHJH~U~~~U~~~~EH-~UU
U C7 C7 C7 U C7 H U ~ C,7 H En ~ C9 H U U C7 U U ~ H H H C7 ~ U ~ U H U H ~ H t9 Eo H U C7 U U C? H ~ ~C ~ ~ H C7 H U
U U C7 H C9 U t9 C7 L7 C7 U C7 U aC U U
U H H ~ ~ H c~ H H C7 U ~ H U C7 H U H
U U U ~ H ,~ U ~ H ~ ~ ~ H H H
L7 ~ C7 t9 U ~C U U H C7 c.9 C7 C~ H U ~ L7 C9 ~,7 H H C9 U U U N H C7 U C7 H C7 H ~ H
H P9 E.., U H U U' acC U ~ ~ C7 U ~ ~' E.~ C~,7 U
U ~ H U U ~ U Z7 C7 C? C7 H U
~ ~ H H U E U E, H U C7 H C9 H H
,c~ ~ H ,~ U L7 ~ H ~ ~ ~ U U t9 U E-i U H U
U U C7 L7 E-i C~ U FC C7 t~ C7 ~ C7 ~ C7 U ~ C-~ U
~r ,c~ E-a C7 C? L9 U c9 C9 ~-i H U H ,c~ .~ U E-~ ~ c7 U L7 U C7 C9 H ~ H H ~ H ~ H L7 ~ U H ~C C7 H ~ t,~ ~ ~ ~ H ~ a; H U C7 C7 ~C ~ C7 'L7 H - L7 H ~ C9 C7 H C9 C7 U H H L7 ~C L7 UHHU, U, ~U~J ,e~UV' ~UC7C7 U"
H ~ H U ~ H U ~ E.., ~ U H ~ C9 H AC U C~ ~
H H ~ H H H U ~ ~ H ~ H C7 U La ~ H ~
E-~ t7 U U C7 ~ U ~ C7 C7 U H U ~ U U H C7 ~C ~ ~C c7 H C~ H H C9 C9 C7 U C7 L7 H H H
U H H H ~ H H ~ H H .~ U ~C ~ H U ~C H
H U U ~C H C3 U U U . C7 ~ U U C7 U U C7 U ~ ~ C9 H H H H ~ ~ H L7 H H H ~ U ~ H
H H H C9 ~C H U c7 AC ~ U U H ~C ~ U
H U AC H C7 U H C7 U U C7 ~ C7 U H C~ H
U U E-a U H L7 ~ H . U ~ C7 U H U U C~ U S.J
UUHUFCC7UUFCHHU~C9UC~HH'H
,c~ (7 H H '~~ H ~ ~ U U ~ L7 E-~ C7 ~C C7 ~ C7 C7 H ~ m4' U U H U H U ~ ~C H U H ~ H U H ~C
H U ~ ~ ~"' ~C ~ H U ~ ~ H ~ ~ ~ ~ C~ U
U C7 L7 H U U H U t.7 L7 C~ ~ C7 H ~C C~
U H U C7 ~ U H U H U H ~C c9 U H H H FC C7 U H Q; ry U H U' H U U U U C7 ~ ~C H KC ~ U' H ~ U ~ ~ C7 L7 H ' H U H H H c7 U H
U U U U ~ ~ H U ~ H U U C~
E-a H [-i U ~C U r.~ U H 'C C~ ~ E-i U H U U
U U H ~C U E-r C7 ~ ~ C7 C~ U ~7 H ~ H U U

'W~ 93/20093 P~.'1'JUS93103Z75 59 ~~3e'~~~sJ
0 0 o a o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1D N O d° O dD N 00 d' O ~D N CO Wit' O 10 N CO d' CO Ov O~ O r1 r1 N N M d' d' lIl In ~ h h CO 00 O~
r1 e-1 r-I N N N N N N N N N N N N N N N N
U C7 ~C U C7 U U C7 H L7 H H C7 U C7 H C~ AC H

U ~ ~C H H C7 H C7 H U ~'C U H C7 U U H N L7 H C7 H U H H ~ C7 H Ch U U t7 ~ U U C7 U ~ U L7 U H H C7 H C7 ~ H L7 U ~ U U
U ~C ~ C9 H H U U d H ~ C7 H ca ~ ~ C7 U U
U U C7 H H U H H U H H H ~ U H ~ H C7 C7 ~C AC ~ H H U H U ~ "U U ~ ~C ~ H U H H
C7 C7 C9 U C7 U ~ ~ H H U ~ C7 U H ;~ ~ H
H ~ ~ U ~ U U L7 H ~C U C9 H U H
H U U H U H H t.7 H U ~ ~ ~ ~ ~ ~ U
H ~ H H H U H U U U
U c7 U C7 Ch U ~ ~ C7 ~ ~ L9 H H H C7 H H H ~ H ~ U H ~ U C? ~ U ~ C7 H U U U
U ~ U tJ H L7 RC U ~ H ~ C7 H t~ ~ ~C C7 H U
U ~C C9 H U H CS H ~ U H U U H H U
C9 H ~C U ~ U ~ C7 H H U H U U ~ ~ U H U
e~ E-a U U AG Ch L7 U ~C H ~ H H i9 L7 V~s"Ug~~~~UHaC~~HHHtU.7UHC~7 H U H C~ H U t~ U H C7' ~ C7 C7 H ~ c7 ~ U C7 H U U C7 H ~ H U U ~ C7 H H U H c9 H ~C
U C7 ~ C7 U ' ~ U ~C ~ U U C7 C9 H
~ ~ ~ c9 ~ C7 H e.~ c7 U H L7 ~ c7 ~ H
H H ~ ~ U ~ U ~ E~-a c.9 ~ H H U
U ~ V t,H~ ~ CU'J C7 L7 rC H H C7 U U Gh U U ~ H
H ~ U L7 H H i9 C7 C.7 H U ~ ~ L.h H H ~ H
~ C9 ~ ~C H H H H H C7 H U H c9 U ~C U c9 C"J ~ U H °~ '~ ~ ~ ~ H U ~ C7 U U H ~~
,~~ChC9E~UC7UU.UHH~CUC9~C~=C
,~ ,c~ ~-c H U U L7 ~ t9 H ~ U H AC U U
U ~ ~ C7 ~ Ei U ~ ~ ~ U H U t9 C7 H H ~ H U C7 ,~ t7 H ~ ~ C7 H H
~ ~ ~ C7 ~ H ,~ H U ,~ U U U U U ~ ,e~ C7 Ur C? C9 L9 H U U ~ rc~ C9 ~ ~ U U U U
t~ ~C H H U H U U U U C7 E-~ H ~ H H
En H H U t7 H H ~C U U H U o4C U H U ~
c9 H Ch C7 ~ H ~ H U ~ ~ ~C C7 U H H c9 H H ~ ~ H U U U c.~ H ~ U H f-a H C~ ~C H H
~ ~ U ~ H E., C7 ~C ~ U U U ~ Ch C7 H U
H C7 U ~ H H t9 U H C7 C7 U U C7 ~ ~ L7 H ~ H H U c9 U H H H H H ~ H H U ~ H
HACU~~H~UHC~7~~U~HHH CU.7 U ~ H U U U H U H U H ~ C7 H U ~ U
H ~ H U C7 ~ ~ ~ c7 U H C7 ~ H ~ H U
H ~ H U ~ H H C7 C7 C7 C7 H ~ U H U U H U
H U C7 U U ~ ~ ,c~ r-'~ H H ,~ ~ ~ cHJ U E-~ ~ C~3 U ~ H H AC ' U C7 c~ U U
H U U U C7 t7 C7 H ~C ~ U U U ~ ~ U C7 C7 H U U H C9 C7 H ~ U U U C7 ~ L7 C7 C7 U H t~
U U ~, E-, H H U U ~ ~ ~ U H ~ H L7 H H
H H ~ H ~ c9 H H ~ ~ ~ C7 H C7 ~ c7 U H
U U U U U L7 U U U (7 H U H
~ H H U H ~C H H ~ ~ H H ~ U
F-s ~ C7 U ~ L.~ H H U ~ U H U H C9 E-~
H U H ~ U H U ~ U U H C7 L7 C7 ~ H C7 C7 U
,cy', H ~ ~ ~ E-i U L~ ~ C7 C7 C~ H Q,' U
H U U U E-~ U H C~ ~C ~C E-~ ~ ~ H C7 ~ C7 VN~ 93/20093 P~.'1'/tJS93/03~75 ~,....,, s 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ 0 0 0 0 O lp N 00 d' O l0 N GO d' O l0 N CO V' O ~.O N CO
O O ri ri N M M ~F d' tf'1 lfl 10 I~ l~ CO 01 B~ O O
MMMMMMMMMMMMMMMMM'd'd' E~ H C7 U ~C H H ~ H C.7 U ~ U ~ t,~ U H H
~ C7 C7 H C9 H U C7 U H c7 H C7 H
U ~ ,~ E:, ,~ U H H C7 ~C t7 H ~ H H
H~H~CH ~U,,U~~HHCU.7~HH

,~ H C7 H ~ U H U U C7 C7 rr~~ U C7 U C7 H ~ U U U U ~ U U H H ~ ~ ~ C7 EH-~ ~ N ~
U,~~,~?H~UUHHC~7~UHUHUHU~C
U H d ~ U H ~ U ~ L7 ~ ~ H ~ ~ H H H H
(7 t.~ C~ C7 H U H U H U H C7 H CH.7 ~ H ~ ~ H ~ C7 C7 H ~ C7 H ~a; H H
U H U ,~ U U ~C U c.7 H U ~ H H ~ H
~K.H7~UH~C-U~H~~~C?CU7HUU~H
U U U H U U U ~C H

H ~ H H C~7 ~ U ~ H ~C (~
~UUU~CC7HUHUC7,U~~H~UU
H H ~ ~ ~ H H ,~ H ,~ H C7 ~ ~ C7 ~C ~
H ~ t7 U H H C7 U U t7 L9 H U U
C H H U U H U ~ U H ~ H H ~ ~ U
C~ t~ H H ~ U ~ U C7 U H C7 H ~ H U
U H H t~ H' C7 ~ H U ~ U U U H ~ U
C7 H U ~ U ~C C7 U C7 H H xC U H U .
H t7 KC U Lh U C7 H U U H (9 U
NH HU H~~~~~~cU7H~~CU,7 H ~ C7 ~ L7 ~ C7 H U
,e~, Ea U ~C ~ U Ch C'J ~ H C7 C9 C7 ~, C7 U ~ H H H C7 ~ H ~ ~ ~ '~C U C7 U C7 ~,7 U H U H C7 U C7 C7 ~ . t7 H ~ U ~ ,~ U a~ H H U H c9 H H C7 U U U U ~., U H U t9 ~ ~C U ~C C7 H
,~ ~ H ~ ~ ~.., ~ U H c7 t? C7 ~ H ~C H
U H ~ H ~y ,c~ H t7 C7 C~ U C9 C7 U ~ H U
~~HU HHHU~~~ E-Ha C7CU7H CU.7 H (g H H ~ AC C9 H ~ t,? c9 C9 ~ U ~ H H
,c~ H U U H U ~ U KC ,e~ H H U H U C7 U ~C
C7 H CJ - H U C7 C9 U ~ U C7 ~ ~ ~ C7 L7 r~ U ~ U' U H ~ U H H H H H C7 ~ ~ C7 H U (~ H t,~ U U H H U U H U H U H
H ~ C7 H C7 ~ ~ Ch H H H H ~ C7 C7 L9 H H
c9 ~ H H U H c7 U U ~ H ~ ~C ~ H U
H U ~ ~ U H H ~ En U H
U U U H Ca t9 C7 ~ H ~: C7 U ~C7 ~ c7 c7 H H (;,~ U U H H H C7 C7 H H C~ H H H
C9 U C7 C.~ H 'H C.9 ~ U ~ L~ H
,.C U ~ L7 ~ ~'C ~ ~ C7 H ~ C~ C7 H U U ~ ~ C7 H ~ H U U ~ C7 L~ ~C C7 ~ H
,cG (~ ~ ~ L7 ~ ~'C H H C7 U H H H H ~
U U ,eC U C7 ~ FC ~C C7 U U r.~ U H C~
H H C~ H H H ~C U U ~ ~ ~ ~ U ~ ~ CU.7 U
H U ~ H L.7 ~ H L7 C7 U C~ C7 ~ U ~ H L7 H H ~ c7 ~C C~ ~ H ~ ~ H H t~ C~
r.~ U H H ~ H H U U H H C7 U U C~ C7 U H H H ~ ~ ~ ~ ~ U ~ H C7 ~ U

WO 93!20093 PCT/US93/03275 r.m..~E

C5 l0 N CO d' O 1D N CO d' O t0 N C0. d' O !G7 N
v-i N N C'1 C7 d" !.n !t! l0 l0 t~ ~ CO C11 01 O r! e-I N
d' d' d' d' ~f' d' d' d' d' d' 'd' d' d' d' d' ttl !!1 !fl 111 H ,~ U ~ ~ U U C7 ~ ~ U KC ~C H H FC C7 E-~
C7 H U U U ~C C7 ~ U L7 C7 U ~ U U C7 U
H c7 C7 c9 ~ ~ C7 H C7 t7 H
H C7 U ~ H ~ ~ C7 U U H C7 ~ U
U U U E, H E., E., c~ U ~ U ~ U H C7 H H
U oQ; H ~' U C7 H Ch C7 ~ C7 t7 U C7 L7 t7 U: U U U ~'C H ~ ~C U
U U U H ~ U U U U H
U U U U (-a U H H C7 t~
C7 U H ~ AC U ~ H FC L9 ~ ~ H H C7 L7 FC
U~UUHHH~H UUC7C7~~C~ UH
~ C7 ~C H H U C7 U ~ ~ C9 KC C7 t~ U ~ U
U H t~ C9 ~ U C7 H H t9 C~ t7 ~ H ~ U
H H H ~ U H C7 H U U ~ H C9 U ~ U U
L7 U H U U t7 C7 ~'C ~ U U U H U L7 U
~ H H H C7 H U U U U H H H ~C U c9 H H
U U C9 U U U U H H H U U C9 C7 ~
H C7 U ~ H ~ H C7 C7 U H U H C7 H ~ H U C7 H o~ ~ H ~ U H ~ H C9 KC H U H C~ U U H
v.C U H C9 U ~.C H t7 H H H U H ~ U U
~ ~ ~ QC ~° e~' U H ~ H U U U U ~C C9 ~ U L7 Ur U (a E-a t~ H U U U H H C7 H U H
H U H U U ~C ~C C9 H U ~ C5 H
U (~ ~C (? U H ~ C7 C7 ~C C7 U e,7 H C7 H
t7 U t7 H C9 H C,7 ~ C7 C7 H ~ H ~ C? U
,y' ~ c'3 U oy H C9 U ~ C9 ~ U H
U U U U H U LU U C9 L.9 U C7 C9 H to U ~ ~ H ~ U H ~ ~ C7 H U U H H H
~ U ~ U ~ ~C ~ U U C9 C7 H H ~ ~ C7 rC
U C7 H H H H U C9 H t,7 H ~ C7 U U U ~C U U ~ U U ~ H U H ~ H ~ U
,~ U H U U U H H U H C9 U U U C7 U C7 Ea U C9 C7 U ~ H U U H U ~ C7 C7 U U ''~ W~
H C? ~ H C9 U (3 H H H C7 ~ t7 U U U
U H ~ ~ H C7 U ~ U ~ H H U U
H ~ U U U H ~C H H U H U U C7 C7 C7 U
U U ~ ~'C C') H U H H FC ~ ~ U ~ H
U ~ U C7 ~ ~ L.~ ~7 t7 U L7 H C7 H ~ U
H U U H E-i H ~ U L7 H H c7 ~ U ~C H H H
U E.a U . ~ ,c~, U C,~ U ~ U H H U U AC H
H [-~ ~ U H C7 Lh L7 oy C7 U C7 U C7 ~C U U' ~ U H ~ ~ ~C "~ U H U U U U
~~HE-r'~U,UL7U~ U~c~7H~UCE7 (.U7 U - ~ C.U7 H H ~ ~ ~ U C7 ~ U ~ C7 C7 ~
'~C U ~ H U U U C9 U C7 U L? t9 H U
U ~ C7 ~ H ~ H H H H L7 ~ ~ L7 C7 C~ H
U E, ,~ H U U !~ H ~ H H U C9 U H ~C ~ E-" H
~ E1 C7 ~ ~ H U H C7 C7 U U H C~ ~ ~ r7 U U
U H H C7 H ~ H U H ~ C9 H c7 H C7 H C~ E-~
U U ,~ ~ H U U H C7 E-~ U ~C U ~ ~ C7 U
~ U L7 U U H H H c7 ~ H H ~ H U ~ ~ ~C ~
U r.>r C7 ~ U ~C H H H C7 ~ C7 H U H C7 ~ H
C;~ C~ H U L~ H U ~ U ~ ~ L~ H C7 U ~ H U
H ~ t7 ~ H C7 H U ~ C7 C7 ~C ~ ~ ~ L7 ~C C7 H U H C7 U H C7 V H H L7 H H H H
U ~ E-~ H ~ C7 H ~ H U C7 H U U C7 a>r U L7 ~ ~ ~C ~ H L7 ~ C7 La C7 E-~ C7 ~ U

WO 93120093 Pf;T/US93/03275 r i>
-~2-O O O O O O O O O O O O O O O O O O O
O °d' O l~ N CO d' O LD N O d' O iL~ N 00 °d' O ~0 N t~7 d' d' tff ltl ~ t~ t~ CO CQ 0~ 0 0 ri r1 N M f"1 1~ tn Uf1 In In tt~ lf7 L4~ lid lf7 lf'1 l11 ~O i0 lD WD ~D l0 H H U,~ ~ H U ~ ~ H H H U C7 ~ ~ U C7 ~ U
H ~ ~ L7 ~ H C7 C9 U C7 H ~ c7 C~ H H ~ U C7 U ~7 U ~C H H H H U U U H H C7 ~C C9 ~C C7 CU.7 H C~ H ~ H C.7 ~ ~C U U ~ ~C U H C9 H
UHC9H~HH~~~cH.7H~UCU7~CC~7H~,?
U ~ ~ C~ AC H U ~ U t9 f') ~ H C7 FC H C9 U
U H U U C7 U U C7 H ~ H L7 U ~ C7 H C7 H
U H H U ~ U C7 H U (,9 U ~ ~ U ~ C7 H H
U ~ U ~ ~ KC C7 ~C ~C C~ C7 ~ U C7 H U U U
U C7 U H C7 ~ ~ C7 U C.7 C9 ~ C7 C7 C7 U
U L7 U ~ H H U C7 H C~ H C7 H L7 U
~7 C7 H H 'H U H E' '~ ~ C~7 ~ ~ U H CU7 C7 ~ C7 U
U E.H., H H H H U C7 C7 U C9 ~ ~ L9 ~ H U ~ U U ~ H C7 L7 H H C7 C7 U U ~ U C7 H H ~ C7 C7 ~ U ~ ~ U C7 U C9 H
H U' U C7 e~, U U U ~ H C7 U U ~C ~ H L7 C7 a~r7t7~UH~U U~HHHHU~H
~HH~U U~U~E°~HU~~.Ut~7 L~7 ~ ~ U H t7 C7 ~ c~ ~ H U ~ C7 U L9 H C% C% H ~ H ~ C7 C9 C~ H U U H U H
~ H r"~ E°° H ~ Z7 H H H H (3 ~ ~~-+ CH-9 H ~ ~ H
U U U H ,c~ H ~cC ~ U U H H
U ,~ H U H U H H U ~ U U H t9 c~ H H C7 H ' E~°a U ~ U t~,7 ~ ~ U ~ H Ch C7 ~C U
H H C7 U ~ H ~ H H U U H U ~C H H H H
U (~ U C7 ~ H ~ U U H L9 ~ t7 H G9 C7 C~ C9 ~ C7 U U C7 U C9 r.~ C7 ~ U U C7 U H C7 H H U H C7 H ~C ~ H H U C7 H ~ ~ U C? . a~
,~C H ~ ,~C U U U ~C U H C7 U H L7 U L7 ~ U
E-i ~ C7 C7 H C7 U C7 ~ H ~ C9 ~ (7 U H H
H H H C? U ~C C7 H U H U H C9 H H ~ H H
(.-i H U U H U C7 H U ~ H C7 ~ t.~ L7 ~C ~ U
~ H ~ ~ U C7 t9 C~ H U H H U ~ ~ H L7 C7 U H H U C7 ~ ~ U U C7 U U U U C7 U
~ ~ E~ H H ~ KC ~ H c7 H ~ C-~ ~ H H H t7 U
C7 H U H U U U H H U H U H U t7 ~C
H U H ~ ~ H C7 C7 H U H U H H H C9 U H
H ~ H C7 ~ C7 C7 ~ H U C7 C7 ~ ~ ~ C7 r~ H
H U K.7 Z7 C7 KC H H Z7 C7 ~ U C7 U U H ~
H ~ H t7 C7 C7 H H L7 H H C7 H U c7 C7 C7 C7 ~ ~ H C7 ~ H U H H ~ ~C H ~ C7 H
U U U H ~ U H t7 U H t7 H U ~ H H L9 U
U U H ~ H ~ ~ ~ H ~ H ~ U U H H H EH-~
U E, U H C7 H H U C7 ~ ~ c7 H U ~ U H
C7 U H ~ H ~C H U U L9 H C3 C7 H ~ H H
~C E., ~ U U ~ U C7 U U ~ C~.7 H U C7 CU.7 H C~.7 C9 C7 U H H ~ c9 ~ ~ ~ ~ ~ ~ ~ U t7 H H
U H H ~ ~ U H ,~ H H H U H c~
C9 ~ ~7 C9 ~ ~ L7 C7 C7 ~ C7 ~ . t9 U
t7 H ~C C~ H U U H ~ H H ~ U ~ H
H t~ H H ~ U t~ H U U ~' E..H., U ~ H ~ H d ~C ~ H
~ rvw I. S.
~H~ ..~rv i . n . .'., . ~; .:x ;. ~ ~.':~: :' . . '~~ ....,:;.. .,.~ _ . .:. .'' ..' .
: :. ~ ~ '. ' , . -;".

W~ 93/20093 PCT/US93/03275 O O O O O O O O O O O O O O O O O O O
N CO d' O lfl N CO d' O tD N CO d° O tD N 00 et' O
d, d, yp ~p h h pp Q1 01 O O e-i N N M M 'd' ~
tp ~D l0 1~ 1G ~0 ~ ~0 l0 l~ h h h h h h h h h U C7 Ch H C7 ~ H U c9 C? ~ c7 U C7 H C7 U H
E-i ~C ~ ~ H H U ~C ~ H c7 U ~C H ~ U C7 H U
~C U C7 H H ~ U U U H ~ U U U U C~ H U
c9 H L9 C7 H H H H c7 H c9 U U U U t7 ~ U U
U (7 H ~7 0Q,' H H H U ~C H U a: H H C7 U H
H U ~ C7 U C7 ~ U U H U H U C7 U U C7 H H
H U C7 H U H C7 ~ C7 H U U C9 C9 ~ ~ U U
U U C7 ~ H C7 ~ (.7 C.9 ~ '~C U ~ ~ ~ U U ~ H
H U U r.~ . ~-a U U [-~ U H U U U ~C U H H
H H H U U U KC H U U U H ~C H c~ c7 H
H C7 ~ C7 ~ L7 U U ~ H C7 ~ H U U ~ L7 AC U
H C.9 C~ C7 H C7 C7 C7 C9 C9 C7 C7 C7 U U C7 ~7 U C7 H E., ~ t' H ~C H ~ ~ C9 H C9 C7 ~'C
U ~ e-~ H C7 H ~ C7 ~ C7 ~ ~ U ~C C7 U
U U C ~ H U ~ C7 (9 f.9 U U C7 U - H C~ U
L9 U H U H C7 ~C L,9 ~ H H U U U H U t9 ,aC U C9 H U H U U U ~ H C9 H U C7 H H
c..'~ r~ rQ,' H C7 H H H U U U ~ U H U H U' U ~C
t9 L7 H U H i9 ~ H H H H U U H H L7 U
H H ~ H U ~ U U C7 ~ Z7 U ~ H H L7 ~ H C7 ~ t9 ~ U ~ ~ ~C C7 H t7 U ~ H U H
U U C7 C9 H U Ei H H U C7 U C7 ~ H U C9 U
U ~, Er H H H U U U L9 ~ L~ H U U H ~ U

U U U U C7 C7 U H H H t9 H H L9 C7 C~ H H U t9 C7 H H U H U H U EU., U U
,v~ C~ ~ H U U H L7 C7 H H ~ C9 C7 U U U H H H U ~7 AC U H C~ C9 t7 H (9 H
U ,~ H U U H L7 U o~ L7 H H H ~ H H H H c9 HU~~'UU~'~'UL~a'C7UC7UUUHC7 U H H ~ ~ H ~ U H ,~ U C7 ~C C~ ~ U U H ~
U H H C.7 ~ U ~ U U U . H ~ H H U ~C H ~ C7 U ,~ ~.., U ,~ (~ t9 U ~ U H ~C U H C9 H H
H H C.9 H H U L7 H ~C U U r7 C~ C7 U
H ~C U H U H H C7 H H H U ~7 H H U U
maC U ~ H ~ ~ H ~ t9 H C7 U ~ U H H
U U H U H U C9 L9 C9 ~ C,9 U U ~ H
H H U U H C7 C7 H C7 ~ U H ~ U U H
H H ~C U L7 ~C HU ~ ~ ~ U U CU,7 H ~ H

UU~ ~~~~~H~~C~7L.7UUU
~ U ~ U U ~ U H,~ ~ C9 H ~C H t7 L7 ~ ~:
cU7LU.~HCU7U~U~~~CH7~U~UH~HLU7 U U U ,~ H C7 H H c7 H C7 H to ~ U H U
U H H c9 ~C H H Ch ~ ~ ~ U ~ U U ~ ~ H
H H U U H ~ H ~ H U. H U U U U
H C7 U H H U U C7 C? C7 H U. U C7 H C7 H
,~ H L9 ~ E-~ ~ C7 ~C c9 AC H H ~C ~ C7 U H H
H U ~ H ~ U ~ H ~ U ~ H ~ U H U U H H
H H U H C7 H U U L9 U U H C7 C.7 t7 U U
~ ~ U U L7 U C7 U Z7 H C7 H H H C7 H H
U C7 H r5; C7 ~ H H C7 H ~ H L7 U° C7 U' U U
H U U t9 H ~ H ~C ~ U U U L7 C? ~ H U U
H H H ~ U U C7 U ~C ~ U H H C~ L7 C7 C7 H U
U C7 H U H U ~ H U C7 ~ U C7 H H
,e~ (,7 C7 C~ ~ U H H U C7 H H H H U U ~ U H
C7 ~ ~C C7 U ~ ~ U H H H U U C7 ~C C7 C7 C~ H
~C ~ H H U ~ C7 U C.~ U ~ U ~ H C7 H H U

77V nit'..,: ....~.-...: . ':- n':~~.. ~..:. ~ ;... :..,.....: ....~. ,. . .
....~..:... .:.... .. .:
WO 93/20093 P(.'T/ZJS93/03275 Win:., ~L,~ .
g o '1~~ ~ -6~4-O O O O O O O O O O O O O O O O O O O
l~ N CO ~1' O t0 N CO d' O ~G N CO d' O 10 N CO d' 4I1 LD ~O r CO W a1 U1 O e-1 ri N N t'') d' d' lCl In r r r r r r r r co co 0o co co 00 °° °°
H ,~ H ~ C9 U H ~ ~ C7 KC ~ U ~ H C7 C7 ~ H ~ H H AC C9 C7 C7 H C9 U ~ C7 C7 H H
H C,9 H H C7 H C~ ~ U ~ U H "~ °~ U
C7 ~ ~ H ~ ~ H ~ C7 U H U ~ U H C9 E.., U C7 C7 U U ~ ~ U U U
~ H ~ H C7 H HH ~ ~ H H C7 U ~C C9 H H
C9 ~ C7 ,cC ~ ~C U
H E~ C9 ~ H C7 H H H ~ ~ ~ CH.7 C~ H H U H
H H ~ ~ ~ U H ~ ~ H ~ H L7 C7 H H ~ H H
~ C7 U' H ~C ~ U H ~ ' U C7 U H H H U H ~ H
H U H H H U C7 H ~C U U U U H c7 H
U ~ H ~ H H H C9 U H ~ ~ H U C7 ~ H
E-~ H H H ~ C7 ~ ~C H U C9 C~ H U ~ :7 H
~ ~r ,e,~ H U C7 H ~' H H C7 U ~C C'~ U
C"1 H U C7 U C7 H H H U C7 H U U H ~
H ~ Lh ~ H C9 H C7 ~ H U U H U c7 ~ U H
U U- H H H ~ H U C9 U U U H C7 U
U U C9 U ,~ ~ ~ U H ~ t7 H ~ U U C7 ~ C7 ~Q', U' o~', H U U . H U E-a C7 H U H ~ H U ~ U' Eo ~ ~ ~ ~ C9 ~ L9 U H ~ H ~C ~ ~ C7 ~C
U U ~ H t'1 ~ U U H C7 C9 H U C7 H E., (~ U ,~ Et ~ ~ ~ C9 U En ~ ~ C~ U C~ C7 ~ ~ ~ ~ ~ U H H H U H ~ H C9 C9 C7 t,~ C7 H H H ~ ~C E-t C7 H ~ ~C
H C7 L9 C7 U AC C9 C~ ~ H ~ ~ U L7 H H ~ ~ H E E-~ H U H H U
H. H ~ H ~., U CJ C7 C7 ~ L7 t7 C7 C7 ~ H U C7 U
U H U ,~ U U H ~ H C9 ~ C7 C9 U U C7 L7 C7 H U U ~ ~ U E., H ~ ~ U C9 ~ ~ U C7 C9 C7 E-W ,c~ ~ H H U U ~C '~ C7 C7 H C7 C7 U f-~ C7 U U U H U U H U ~C C7 U U C7 U
,~ ~ U ~ H ,~ U U U ~ ~C ~ U ~ ~ C7 C7 U
U H H H E-i H C9 H U U H U
U H KC U H H C7 C~ E-i L7 C7 ~ U H U ~ ~ C~
;~ H C9 C9 C7 U ~ C°a '~ ~ ~ H H En ~ ~C ~ _ H H U U ~ ~ H ~ ~ U C7 ~ H H ~ HH C7 ~ H
U ,~ U ~ H ~C U ~ ~ U C7 E-~ ~ ~ U H ~ H U
U ~ V ~ ~ U ~ ~ ~ C.7 CU7 ~cC ~ H C) U H C'J ~ E' U U H U U H U U
U H H CH7 LU7 ~ U H C~7 H H U H H ~ U ~ U
U E-Ha E-~i r.C C7 C7 L7 H Ch H H H C7 C7 C7 U ~ U
~, . ,,~ H C7 ~ Ca ~ H ~ C~ H ~ ~ ~C ~ H H U H
U H H C9 U FC H H ~ U H U C7 U U ~~
U ~ C7 H ~ C7 U H H H H H ~C ~ U H H H C7 t~ C7 H H H H H H ~ H ~ C7 U U H H U ~ C7 E., U U ~ H ~ ~ ~ ~ H ~ ~ U ~ ~ U H
C7 H ~C H C7 U H H H ~C c.~ C7 ~
H ,~ ~ ~, H C7 C7 ~ H H H H U ~ C7 H U U ~ C9 H C7 H U U ~ ~C
C7 H U ~ U t7 C7 H ~ ~ ~ ~C H U C7 L7 U ~ ~C H H H ~ H U aa;; H ~C ~ ~ U
H H H H C7 ~C ~ H U ~C U ~ U U ~ L7 H U ~ ~ H ~ ~C ~ E' H U C7 U ~ U ~C C7 UU~UUHHUUUUHUCH7C~.7UUH~C

VV~ 93/20093 PC.'T/US93/03275 O to tV 00 'd' O O
~p pp O1 O O
CO 00 CO d70 OQ ~1 01 E~-,c.H7~~HH

U

E~-s H H ~ U H
U H H ~ U
~ N ~ H H
~.U., U ~ ~ U
G
U CU'J H H ~ ~
~ ~C ~C
U U ~U., E ~ H Pn ~U
~ it H ,~ C7 ~C7 U H °

U ~ U ~ t~7 CH7 ~° ~--t G ~
U ~ H H C7 H -~ 't3 -~-t !~ .~a U ~ H ~ H H O cn ~ ~~~i tn ~o ~ ~
~, r~ U a~ +~ ~ ~ a c~7UUHV~ Z~ ~..~ O ~ ~ .
H ~ ~ C7 U H ' L7 W c~ t!7 ~~ ~ ~ O °~
a-~t H eh O tlW S3. O
~ H U ~ H H ~ '~N ~~~ w z z W ~~.H
H ~ ~ ,~ H H cn ~ it W c7 a, ,-a c~ ~ cn U 47 U H H x x D O 9~ ~C ~~ ~ O H W
U U ~C U U H GU E-~ ~ W --l H U W En H ~C C7 U H ~ O C9 W ~ O w tI~
U ~ U H H tsa W ~ 4~a Pa W H ~ H a C7 UJ
U W ~ H O a W W z ~ !~4 H
c.H7 H ~ U H Z ~ a H c~ E-r ~ x t~ W ~ O H
H H C7 H O W ____ U H t C7 H H H a ~ c~ U D ~W-7 a0, H ~ >U-t ~ ~., ~ wr v aws t!~ ~ x ~ W O
H ~ U C7 H U H H '-' -,..~ .~ .v '-' C7 U U C7 H ~ ~ °~
c.'~~ U ~ U ~ E-~~ ~ cu ~,~1~ ts:r r r ;s. a '~ ~..~ ~. 1 ..".a.,..: . ....ajs::, .,.o..;~.~, r .Pn.,.~.' .1 .n ..n ~ . , ...." . ... ..
r . .. . , . .. ... . ~. ...,.... n . ......
~~ 93/~0~193 PC'T/US93/03275 4..'l"~c~~
o in o .-a ~ iu ~ ~ ~a o a ~ ~ ~ ' a a o ~

a, a ~ n c aa o in m , a a ~ H
~

c~ w c~ a c~
~

o o s~ ~ ~ in ~ ~. cn s~ ?P ~ o o r E..~ s~ H ~ ~ x ~ ~ a r, ~

o ~ ~ ~ oar ~ ~ o 07 ~.1 fi5 ri Pi N N i!> N Q1 r-~1 .Ci to N

fJ~ ~ J et' H ' (n V.1 ~ a (!) H H
[~ r.'~

ri ~-1 o r-I l~ UI r-1 r~ t~ r-1 fd '?~ N
o d' a a ~ ~ ~u~ H ~ ~ ~ ~~ ~ a i.~ N ~ ~ ~

.~ r-o ~ t~ i t W H ~ ~ 1. C7 H L7 L7 n ~ ~ ~

-i ~ ~a ~ o ~ GL ~ o.P o ~ v~
o . r tU C: ~ S-I tll fa rd ~1 fU N ?h ~ o ~
~

. a w tea. ~ c~ ~ a m~ a E

~ UI ?, ilJ 6d tT r-I N 5'".,10 l~
fn ri ~t Vl ~"~ ~ ?~ ~ Si ti1 .~ tW -I CO
T if o i , o~ G4 ~ x U' a ~ ~ ~ U ~C '~ r1 ", N r-'~

C S~a O

e--1 'fir aI Q3 U7 N a a w~ ~ a a cn ~~ H a a ..a ~, ~a o ~ ~a in ~cn ~ ~ '~ o H

~, a e ~n n ~

o ~ ~ a~ ~, ~ in a, en ~, o H a, ,-, ~ ,~ P-~ .-~ ra .o -P~ cmn o H , C7 Lh W e,9 H C7 H x ~ a C7 E-~ t~ ~

~
u~

N r~ rtf ~ N N r0 r-t ~1'~ ~

U H a en Z7 ~ > ~ U to c!5 a c.7 ~ o -j in ~ s~ ~ b ~ ~, it ~ tr o o G~td' V~ ?~ ftf ~-1 (a ri ill r-1 r-~i Q5 S-1 CO
O o ~, ,..~ :% E"~ J ~, ~ V' ~ a N ~..~

U Ir S-a O ~ O ~ ~ ~ ~ v A
~

i t ?~
o n .
-I

W ~ t G ~ ~ H x ~ C7 ~ ~C U
l)i u ~ ~

pt~,.,,~ r-~ ~ O t1 ~ ~ o o O

i r-i (0 N ~.1 N CV ?Y ~ M ~ rt3 ftf -' O

en", c~ ~ a w ~ a ~ w ~ w ~ >
H ~n -.rtS .N LT ~ i~ f-1 S..i ~ O ?, N
in . 1 ~ ~-I C ~ ?, ~ r-~ .C. r'-i -i Q1 - In V' . r ~ ~c . ~ ~ in c~ a, c~ x a x ~ H ~o ~
~

W~ 93!20093 PCT/LJ~93/0327~
~ ~ t~, cn ~ v ~ .N ts~ s~, v o o S.a v ~.1 -I H ' x ~d' ~I fly 4i .'~a C~J H (J c~ ~ ~ W
m N ~.

~ ftj ~ ~a ~ ~ ~ ?~ ~ ~1 I l11 tn - r-1 (n f-i v r-1 v r~ v .C, v Ul C'1 ~c ~ ~N a ~ a c~ aM H

an ~ ~ i~ v ~ t~ i~ ~ ~ 0 o o ' '> ,-i fd '.~, "C.' v (J1 N r! N Sa t~ ~c y ~ ~ ~ wN a ~ ~c ~ aM a. H
a ~

a, ~ r, v s~ .a~ m o s~ t~
~ ~n cn H
r~

Ch N ~ H ~ H V) ~ x W ~
N

~ ~ ?W i ~ ~ ' ~0 ~

~ r! r-i i!) U7 w r..~ ~ r-1 v v O
N ' a M

c,~ ~c C~ Lla cn ~ a ~ c~ ~ ~
N M

~ N U1 ~ ~ N ~ ~ ~ o u7 ~

~ ~ Pn ',fitri r-1 v r1 v-I cn U7 C'1 .--I ' a,' ,~ ~ a x C7 :a x U ~c r! ~ f-1 ~-I O ~-I ~r V7 O ~

ri f~f ~t tC ~-1 .~i v ~ ?t UI ri ~ lIl M

W C7 ~ ~ ~ ~ H cn ~ H ~ ~C
N ch ~ ~ ,~ ~ V~ ~ ~a t~ ~ ~1.
~5 ~

r-i v ?W n en ?~
~r H x C7 ~ C~ En U ~ ~ a N ~

~ ~ ~ N ~ O ~ o <C v 9a ~ v t~
c~

., ~, id! r~i r-i v r-,i .C ?, co e~ a ~ N a ~'' U ~ ~ N ~ H U7 C7 ~
ch ., ~ is ~ tn t~ r~ ~ to u~ ~
~

C ~ N r-1 .i,'"~-I .G r! U1 r1 ~.1 ' r-( t31 ' ' C r-I
' c 4;
M

. ~ ~ U H d, H .~.. ~ ~ ' , ~ N N ~ .

c~ v ~ ~ ~ tn ~.-r o c~

U7 ~14'~1r-I .~. t'd Qa 'fit PLS v-I Ia f(~
~ r'I

a ~ a H H a a.a U r c7 t~ M

~ ~ o ~ ~ ~ v v UW ~ ~ is ~ .c1 f-I N r1 v .~: ~'r 'r r~1 l~ v ~' N

a c~ a c~ w a N M

~-1 ~ ~ ~ ~r O

,..d ,~ ~ '-~d WO .~ ~ v tt1 ttl N ~.a d~ ~C

H N H c7 ~ c'~ H a ~ ~ '~
N M

v ~, ~ ~a o ~ ~ ~n v o ~ ~ .~ v u~

r, ,--, ~., ~a x ~ ,-, v ~, u~ v r, ~n (~ r., U' ~ ~a C7 ~ N H a H ~ ~. H
M

N s~c~ ~ ~ v m oo v co ~n ~ ~o wN w ~ a a ~N ~ a ~ x N cn r~ ~ ~ ~, ~ mn ~ +~ s~ v En ~ -,~ ~ v ~a ~ v ~-, r, v v N o ~ ~ ,~ tn ~ L7 U1 U' C7 :~ CF7 H
N M

WO 93/20093 PC.'TIUS93/03~75 ~ g~t~a~~;
~ o O S-r O fU O N N N td ~ O
O O
vD

O ~ r-1 N Pr AC ~
~

y~ e p H H H H ~-l a U
p .a d' r~ ~-1 ~ :y r1 ~ In ~ In ' J (a (u t~

f(~ r~ ?~ r r-I r-1 ftS ~f ~I Q "r ~...7 In r1 i ' Ol ~ ~l ' ' ' ..~ ~ H U H U J' H .
~, e1 G N O 1-r ~ N ?~ VJ O
O

O N ,.-1 N O -rt ~ >'' f.a N
~..~ .- ri U~ H
a ~ ~ ~ c~ a x c~ a ~, a~

o ~ ~ ~~ ~ o m a~ rau, o, ~ a a a a w ~~ ~ a .

~ n ~n . c a w x ~ ~ a~ w cn c~
~

~ ~n ~ ~ ~ rs., ~ H~, a c~ a ~o ~ n~ ~ ~ ~a ~ ~ ~ N No N ? 1~
?

N ri n,7 r-i r-i N 1r r-1 .~ ~ ~ q ri c'7~ H a a icy ' ~ ~ ~ w ~ ,~ ,~ t7 ~, d ~ ~ ~ u~ s~ ~ ~r rn o u, o w H ~ a~ ~ D H ~ W m 4L ~
~

~ ~ N :7 ~ o ~1 ~ ~r ~ ~ S-d O O

r-i r-I ~.1 A) N r1 .C O N
N t1' N

,~ ~ a ~ . w c~ a c7 a, a cn d, ~ ~n o ~, ~ tr ~' a' ~ ; m ~ ~ w ~n ~

~ ~ H C a ~
~ t ~ ~ H ~ w ,~ W w.. .

~ ~ ~ ~ ~ N
~ ' ~ ~ ~ ~ ~

cn o-.~ H Er wn a w c7 cn W H _ ~ e.n ~ ~~ ~ ~ ~ ~ um ' rt ~ ~ ~ o .
c ~ ~~ ~ a a ~ w~ a, ~ ~ c~

~.,o ~ ~ ~ ~ ~o ~ ~ ~ o c H c ~ ~ C7 pr r-~ C7 ~ ~ ~ Ei E-~
d' -~ N ri is ~ ?, t!1 r-d Ul r-I ~ o N ~ ?, cd l~a N
~"~ ~-i ~C a C7 n ~C w H W ~ ~ ~ W o r.-~r O O ~ ~ ?i N ~ ~ 1-1 ~.1 7a t3~
O O

r..~ y~-i ~1 ri ri ''f U7 ~ .L~ .~i ra ~-d Lf1 f~1 ~

H ~ ~ a c~ a ~ a H ~ c ~ ~n i ctf ~ ~1r ~ ~ ~.d N ~S1 ~ ~-t 1 1t1 lf7 Lc1 -1 ' , r-f r-1 N Ql .C .C -W -1 N QI r - l0 eP
~

t ~c c~ ~ a w H x H a v~ c~
~, ~, ~ ~, ~",~VV~ 93/20093 PCl'/US93/03z75 ~

r-1 .~: aI ~3 r'~ f-1 e~-I r1 r1 d' N ~ a w v~ a~ H ~ H ~ c~~ ~

v ~, ~ o ~n H c~ ~ H c~ a, a H H ~ c~ H c~
~

~ o o as ~ s~ ~ ~ .a~ s~ ~ ~a c~ o a~
~

w w H cn a t~ ~ E~ ~ cn H
~ ' ~

~a, .-~ a. cu s~ o a. ~ ~ o ~ ~n r-1 U) f~ ~-1 .~" .~''..~-1 r-1 N ?W d UJ l0 Ac ~ ~ a~ ~ w c~ ~ a a~
u~ ~

~ ~ ! ~d ~ ~ C tT ?~ I-I U1 O

H ',w ~'1 ~i 01 UI S-1 r1 ~ -ri f~
N O

H . c~ ~ H r~ a ~ ~ ~ cn x .o ~.

roe, ~ ~ ~ ~ ~~, o a ~ ~ ~ ~ ~ c~ ~ r~ a ~ c~
c~

~ ~ ~ ~o ~ ~ rn ~ oo ~a o ~ u~ a~ ~ ~ o ~, a~

H c~ AC a cn ~aC ~ ~ ~ a r. cn w ~

A, ~n ~-, o o ~ ~ s.~ v ~n ~ ~ o ~ ~n b ~,~, ~ H ea a c~~ ~ w a ~ ~c~ H .

~, ~+ m ~ ~a .t~ ~ ~ ~t ~ t~ o ~ o ~ ~ -~ .c >. nr v~ m r-, o co ~ c~ cn x ~ H ~ ~ ~ ~ t9 H ~.
w ~

~

~ -i ~ N fty .L". ~ 9r N ~ .t:
.~j 0~
r ~ a ~ ~ ~ tn U t!~ W '' .
eD es~

N o 3.~ .~a ~ ~ b.a N ~ ~
o ' ~ 5 ~D '>r -C. 9I ft'! U7 W ri .C. H
~ M -I

.. ,~,,''U 10 EI ~rr ,J q~ U7 H A.1 d.
I~

'--i ~ N ~ N
~

, ", C, - ~ U1 ~ U1 ~ sy , N fA
d' H ~ C7 a Ei x t!? ,~
t0 h r-1 :~ ~
~ o jj~ CO o ~fJ Q~ r~ -rW0 r '3y tU r1 ~..1 .Ii -i d w ~ u~ ~ x c~ v ~ ca ~ w ~ ~.

' n a ~ a c~ ~ a N ~, ca ~
c ~

it ~ a~ ~ ~ ~ ~
o o ~, ~ ~ .~ a~ as a ~ ~ a ~ U H Q, cn a a cn c7 ~ ~o , ~ ~ H ~ N .C: .C r-i N t0 r1 r1 N o ~n ~ c~ a ~ ~n ~ w H a ~ H c~
~

Vd°~ 93/20093 PCI'/1J~93/03275 _-~p_ c ~c~~r' ''~ ~' n ~

r. o ~ te o ., o p p a e P c~ cn ~ x w ~ N ~
, , n co c o ~ ~~ ~ +~ o ~ ~~ ~ ~ c -, ,~ ,o v v v v ~ ~s v N v ~ o, c~ ~ ~ a ~ a cn ~o 0 00 ~
~

.- ~ ~ ?~ cd v ~ ~ N a a ' ~

c en ~ a ~ a w ~n n co o~

N ~ ~ ~ ~~ o N o ~ N

m v ~N

~ H ~ ~ t7 a ~ ~ a o. c9 c7 ~

0 0 o m o s~ c o v a, s~ ~ ~

y.d (7p r-1 ?e tla v U? .L; N v J's vO

~, c~ c~ a ~ ~ ~ ~o s~ ~ v~ a c~
m ~ ~ ~ N ~ n. ~ o >, ~ s~ ~
~n ~n v ~ ~ v ~ v N v r~ ~ v v ~n o, c~

~ ~ ~ rn x a AC cn s~ c,7 cn cn rn r,. a~

O ~ ~ pa N ~ ~ O O v ~ O
O O '' I v .-I V! -rl ~.I v '~.t W-d .~ v N
x-i O

- a ca ~ x ~ a w w w a oo ~o ~ ~ ~ vu, ~ ~ N V ~,m ~ s~

,o v P, :~N v v ~, ~ ~o v v v cn t-~ a a a H t9 a oo 0.

.4a ~ b ~o G N ~ ~ i..~o v v ~ r, ~ ~, ~ v .o v v ~r N

,cc ~ a ~ C9 c.~ a Er a E~ a a co o, u~ ~, o a. ~., o o ~n o ~ ~ s~ o ~.
' ~

r1 I~ v UI ri ~1 v lCl v v v ., ~-d r-1 .~ M

x ~, a ~ C7 ~ a ~ a cn cn H tz. C7 .
o~

~a ~ s~ o o v v ~ a~
o~

~ a ur w ~ t~ t~ H cn ~ c~ ~n rn ~ co a~ oa o ~ ~ ~ ~ o ~ ~ ~ o ~n ~

~ N N v :L' v .t"..v ,C,''v r~ r-i O ~

, a cn w cn H a ~ a c~
, on m ~ ~ ~ o cn o ra ~ ~ ~ s~
o o ~I v v ~1 ri ri r1 U7 Ul ri N v N O cn a E, cn cn ~,, x c7 ~ FC ~ ~7 co o.

o ~ o ~ s~ o ~a o a~ ~ ~n ~ o ~n ~ ~ ~ ~o ~ a a ~ ~ ~
o o ~ ~. ~ .~.3 ~r ~a ~ ~ ua ~ N
~ o o ., v ~ N v .~ ~ v .~ N x ~ ~

H~ H ~ H ~c ~~ a N ~ o v ~ ~ ,~ ~ ,-~ a~ v v ~r ~ ~

E ~ x ~ C7 w H E~ L7 Ei a tn Sr ", ~ co 'WC~ 93/20093 P~1'/tJS93103275 ., h Q O

Sl1 (~ rtf ~ U? fd !~ f~ ~ f-1 Ul ~cY N

r-I N .~, ~ ~ a ~ ~ a a a ~ ~ t7 en ~

~ ~

~, ~n w ~ ~ a~ ~ ~, o o ~ ~ b u~ ~

r1 h .L,''O r-4 .C, N O f-W -I tIJ
O ~

a, U~ C7 W ~ tJ~ ~ W ~ C9 ~

o!a ~ ~ N ~ N ~ O ~ ~ O
O ~1 t~

5y Q~ !t~ v--1 r--1 '.~f O ~.1 tU (17 S-1 (tf ~-i , aa, ~ H ca a en~~w a a w a ~ ~ ~

~ r N N . .~i N .C; r- ,C ~ ~- ?, -I O i 1 a O

, a H ~ H H~ a, cn ~

0 o ~.1 ~ N fir .b~ ~1 O ~ O ~ ~ .S', ' O ~ O . U) al .C ~-1 O ra t0 ~ N
' ~ O

a ~ cn a~, ~ ~ H c~ a~, ~ a ~, ~
~, ~ ~ ~, ~ ~ ~ ?~ ~ ~
M a ' a , ~ ~ H n ~ a c~ ~ a~, on a c o o ' UI UJ ~ :7 ~ ~".. :~ U) ~ r-1 '...!' Yyq O t!7 M

, 'a't '~'r ~I r1 ~l U1 r1 '~~o r1 fIf ri r-i i'- O r1 ~a a~ r a w c~ ~ ~ c~ a c~ ~
a ~ ~' ~

s~~r:~ ~ ~ rn ~~ m o O ~ ~ o-s N ~.s N ?, ~ o cn H~ ~ c~ a ~ cn~ a w x a ~ n a ~ a H ~ H ~cC ~ e:a W H ~
r-1 r-1 N ~ ~ ~ N ~ ~ ~ cn N
43 to .-~ o~

-t va -~ -~a ~ a~ ~ ~, .~
o s~

~ a a ~~ ~ x x c~ cn~, ~ a ~

cn o cu ~ ~ .u o o ~ ~
M ~

O ~ O -~ P-1 r-I tl~ r-I ~I r-1 3-1 N
O r1 v~ ~ a x c~~ ~ ~ ca ~ ~~ ~ ~

~ ~

1 tf1 O :~. S-1 ~ ''.3':3 P"~ ~r t13 f1 ~ d N

. S-i O ~, !.-I N N fd Il1 r-1 Ql ri Q1 1fl O ri corn w a H H a~ a ~ ~ ~ v~.~ c~

o a s~ m .-, o tr -s~ o a, o ~ ~r u~

rd r--o a~ ~ ~i ~ ~a v s-a co o ~ C7 tn ~.-l ~ W ~ ~ W H W c.~ ~
0~ ~

~ Ui N ~ f.~ ~-I :~ ~ ~. ~' ~ i It5 ~
t~

, r- r ~ -~ ~ ~ a~ .~ a~ ~ m ~ d o, o C7 x t--mn~ ~ H a C7 ~ ~ ~7 ~

t~ N ~ ~ O ~ ~J -8~ o ~ ~ ~ o~

~ ~ N U~ ~ c~ ~ v rt ~ r-c rtf o o a ~ tf~ aC C~ ~ ~ ~ C7 ~ C~
~ ~

r-i f..1 ~ f!1 1-1 ~ ~ ~
~-1 ~
N ~

r-I N
~ a ~ C

C ~ ~ H c a E ~ cn c~ cra 7 n -~ ,-~

W~ 9312410'3 ~'CT/LJS93/03275 _-!

~y ~ u~ ~ t~ ~ cn ~ ~ m ~ d' o oo ~" r-1 '~ .~." .~' Q1 W QI i-1 >t ri (U
N N

v a~ H H a x a~ ~ a c~

-t Ud " ~ S-i r-i <n ?W ~ f..1 ~' N 1 r-s Clv r -r-I .. rl N ft! '?t r-1 ~'., LU ~ U7 b r4 N N a a x c~ x~+ ~ ~ a c~ H~ cn o ~

J ~ N ~ '~ Q U1 ~ ?.~ C3~ ~ N
~ M ~

- ~ ri ~ ~ ~1 ~ ~ .G ~--1 r-~
(~ N M

~ a ~, H a,~ w~ a .- c~ H ~~ ~ a ~ ~ '~ .~r .~ ft7 ~-1 '~' UI ~: ~ ~-1 ~ lD '~' ~-i ' N
"

.. . r-1 ra N r1 ~''.. ~ 9r r-I .f O,I
r-I ri N .r ~~, ~c c~ c~ a ~~ H a v ~ H~ cn u~ o ~ o a~ s~ O ~ crf ~ ~ t~
a~ tr ~r oo ~

'?y U7 r-t ~ ~' ~ 1~ C9 r-i e-1 N

a ~ C7 G~ W E-~ ~ -~ ~ ~ H C7 H ~
e-I

'~ "~' ~ ~ ~ P~ G ~ ~~I r'1 ~, '~
d1 I~

Q? 43 0--1 Q? N r1 Tn ri 'fir td r4 ri r1 N

a '~ C~ a s.a ~cC ~C U' E-d a (~ C~
ri ~

' P~ ~ O .~, ~1 ~-1 ~ ~ ~ :~ ~-1 ~ ~ V1 T'~ Ca1 , [)y r-t t~ t11 r-I .~," ~ ?~ .~ Q) .r.
y N N

C7 ~'' a'-~ a ~y' C7 E~ U a t~ ~l E-~
r-4 ri ~ ~

N a-~ tJd ~ ~.1 ~ ~ ~ O b-1 N O

(~ t" rhy fd -r~yq~Jr-1 Q) r1 r~i U7 ~1 N
M

a " a a a c7 cn C~ c7 ~ w cn E~ ,-~ ~

o :~ U~ f~ d3 o L1i ~ r! 'j.."o f-1 ~ l0 3~.i ~
'd' N

N ~-t ~ r'~y U1 N 'r~~aU1 r-1 B r1 ~1 .G
IV M

,~~, ,~ a ~ ~ E.,~ ~ ~ a c~ a~ H

~ ~ ~

IIJ g? :9 ~y ~ ~ ~ tt1 U1 ~ ~ ~ ~ M
t~

a; N Zn ~r rW -1 f-! U1 N M

;~ ~, c~ c~ ~ ~' ~ ,~ a ~
~, ~ ~ ~ ~ o ~ ~ u~
~
c~

ri Q) '?v r-I N .i; U7 N

a c~ ~ ~ c~ a a c~ rn w ,~ ~ ,~

~

(fj r-~ ''J~ ~ N ~ '?t .G r1 ((S
N N

W '~ '~ ~-a ,"a c~ tn tn a H t~ a ~ ~a o o N ~ O ' ~ ~ ", ~ ~e ~ .L: .C: M yy ,~ N N

x a H~ a ~ w H ~.~ a ~ ~

r-d ri N Ca .t: ri Ul ~,' -r~ ri ~d AI e-i N M

ca ~ cn . H ~ ~C ~ x ca w a H ,-t ~

O O O

Ua ~-t ~ ~ ~1 ~ i~ fa ~ ~-1 (~3 ~, ('~ tr1 M

,~ ~ U1 r-1 .L; ri N N r1 fn N ri ~-!
r-i M

a ~,~ ~ c~ H

r-1 I In ~e 1d ~ r'.'~ ~" r-i r1 ~ ~ N

- ,~ a a ~c c~ H c. H ~ a ~, iWO 93120093 PC~'/US93/i93275 <,,.,..,.

Q o 0 tn ~ U7 !I~ G ~.a 3.a ~ ~ N C
Wit-N vD tT ' O ~ ?~ r-1 ~r .G Ul N tf1 N
er a~ ~ r a a a~ c~ H H ~ a~' a ~

~ ~ -~1 c ? '~a Vl .i: ~ ~ N
. f ~1' t , r t ~ a ~ E~ a a ~ H ~
P- ~, ~C ~ a ~
t ~
-~ ~-, o , y o r1 O ~ ~ N
h c i ~~ w ~ a a a ~~ ~ ~ --cn o ~ ~ ~ C ~
o , .

~ ~ ~ ~~; Ho c~ H u~ a~ H x H

o ~' ~ ~ ~ ~ ~
' ,~ a n~ t ~ H [7 ~, r 7 ri u7 ~ In f ~ o S fO :~ :~ ~1 O U7 o r-1 ~r i M

~ L .1 ~d - H Q) r1 .~i ~-I -r1 3d r-i QI ' e1' !!1 r-i M ~

H e-i ~ . ~' a H ~ H f~ x ~ ~i C7 ~ ~ ~ ~ p ~ ~~; ~ o ~ ~ ~~, ~ N ~ ~ oM

.o o ~ ~ ~ o ~r c~ a.~ ~, H ~ ~ a~ ~ a ~ 0 o N N ~
fir o0 s.~ ~ s~
~, H ~, ,~ ~,, ~, x H ~n a, a c~ H
~., ,-~

~ ~ ~ o ~ 5.~ ~ ~a a o ~ a, ~, ~
o co 0 0 ~ ~~ o a~ o .o w~ ~ m ,.a ~ ~ ~ a cn a H a w ~C c~
~ ~

~ ~

o~

~ ~, ,~ H ~', U1 H C'! ~ a ~ ~
r-i H

O Q 0 ...
~ ~ C ~i ~-1 '~ f~ :~ ~ ~ t~1 :3 t"'7 r-1 ~ -i H $1 Dl ri e-i Q1 .~, O .Ci O
G~ M ice er lf1 . a H a ~~ a , ~
~ o 0 v N ~a N ~ ~ ~ N ~ ,-i Wr, M er Pa H c:4 ~, cn C~ a cn KC c7 L~ a ~
~ ~

~ ~ ~ a~ ~ ~ ~ ~ o ~~ ~~ '"

ri ri .t"..U7 f~ O ri ri td .4 O
M d' ' H ~4'r H ~ A:i 'J ~ C7 L7 J H UJ
~-I ri ri '-i Ul O
~ ~ t'~

e- ~ N U7 I g) f~ l~ ~ ~-1 .~ C1 r-- ~1' a c~ cn cn~ ~ c~ a a ~~ a w o o N U1 ~ C ~ C C N ~
~

. .C
' ~ ~ ~ a ~r x v ~~, ~~ w c~
, N C d~ ~ ~ ~ C ~ tn S~ ~ e--i a~ ~ o c~

<r r-, ~ rt~ .C N tr N ~ ?~ N 47 rt3 ~ ~r, M

, ~ ~, ~ ~, ~~ ~ ~ a cn a~
a~, ., ::. . . _ . - : . .... . . _._,. -: " .:..- ;;:,: .:. . - . : ~v :;. . , , ,;
;~.,~y , ....... ,". .:r~.. ....~ ........ ... .... .. ... .... , .. , ....., .... ,:

W~ 93/20093 PC.T/t~S93/03~75 ~ee~
t ::; .~~'~~,1 Q O
far ~ f-! ~r ~-I ~T ~-1 ~..~ t7~ ~-1 O
O ~

r~t N r-i N r-d ~".. f-~ ?~ ?WO '~a O f-a ~ t!

C7 ~ C7 tt~ C7 Ea ~ H H ~ ~ a r-I

~ ~

fa ta ~ ~ ~ tr, 1~ i3.~ N fa .a~
.-n o~

4"' i r-I v r-4 f,.~ r-4 N ,--1 .ti IU (~f ~-1 u7 l0 . H c~ a c~~ ~ c~ ~ H H~
H

n td fa O O o N 0,1 Q, N ?a fa u7 5r ~e-1 M

td ~e ~ ~--~ N mn -~ ~ N ~ ~
~ ~ r ,~,~ ~ ~ ~, ,~ ~~, ._ H ~ a c~~
x -i ~ N f.1 Q7 ~'.. ~ ~ (U N f~ ?e N
" C3~
~ e9' t0 r ". ?~ C ~ ~ S.W ~ .C ~ ?m .-i r ~ a a . H c~ ~ c~ f~ a H
~ H ~

~ 0 o fa r~, ~a N N ~ a, s~ a, s~
~o ~o a ~ N ~ N a~

~ ~ H~ ~ ~ a ~ ro rtf ~ f~ ~ r t~
tn ~

N ~-.~ ~, ,~ ~ a~ r-, ~ ~ x f.
u, a ~ ca a ~ ~ a c~ c~ f~ H c~ , ~ ~

o o Q? ~ ~ N ~ t2, r"~ Cla N ~ :3 r-i Q~

-i ~, ri fa ctf N ?, N N
e0 'D

~, ,~ ~ a ~~, H ~ ~ a ~,~ a ~ ~ ~
o ~ N UJ

~ te ~' ,, a ,. ,,~ ,~ H f~ cn a ~ ~ ~
.., a a-a ~

~ O dl N W-'~ o f~.. ~ O :3 fb N
N O
~D d~

r-f .,.j ~ ~., r-t at f-a ~ N r vp ~ a~ ~ ~ ~ ~ f~,~,c~ ~ f~ c~

~ ~ ~ ~ s~ ~ ~ ~ ~ ~ ~ >T
o~ ~~

,?y Q? r-i r1 N r-I N WD r-i 43 tiS
ttl ~

a ~n H ~ ~ ~ c~ ~ a c~ ~n ~
~

~ o ~ o ~ ~ s~ s~ o ~ ro ~~ ~r -a ~., ~ .~ v ~ r.a .~ rd ra ~ s~
u~ ~ a ~ f~ t7 H va H c7 E-~ a ~ c~ ~
~

'~ ~

~ ~ ~ N o ~ ~ ~ ~ ~ ~ b ~

~ . c ~ n a, H ~ a ca t,~ cn H cn ~

Q N ~ ~ N o N ~ ~ ~ ~ N
~~ O O
N

f ~., r-i r-1 ,'~ f-1 ?r ~-1 r1 r-~ r-1 r'1 ~ ~5 l0 r ,. a ca c~ c~ w r~ ~c H c~ a-a x ~ ~-, ~ ~

~ ~ ~

s~ m s~ o ~a N ~ fa ~ f~ v o ~r ~, ~, N ra ~, i~ ~ ?, N .~ .~ N .~ 5-~ r m ~

~ ~~ H w ~ a a~, H H ~ f~ w~

ci~ ~ ~ fa s~ f~ 'a o ~ ~ f~ f-a N r ~7 ' u~

r1 ~ -.-1 d? N N ~..~ r1 N r1 ?v N
Ifl i0 ~C tn :~ (O ~ ~ ~ H ~C C:~ H
r-i .-i f~ f; ~ ~ O ~ ~ t1 f~ N Y~
N i0 o0 Q3 N ?~ tU ~ ~ ~ N ~o N -.-I N
tP5 cn ~ a a c~ c~ c~
~

tW0 93/20093 PCT/1JS93/03275 <. ., o ro , ~ ~ "fi ?~ H N U3 .L"'..N (n r ' CO -I1 Ov ~ t ~ r a ~ ~ a H u'3 ~ N ~

N C9 ~

N ~ ~ !-1 -1 .1-~ ~..1 ~ r-i tn U~
N Itl f' ~ O .~ N td N O N t~ ~ ?~
t~ to x , a W u7 ~ ~ tJ~ a ~ a ~ U
C~ ~ ~

a o N cn O N V O ~ ~ 0 0 t~ "

~ ~ ~ r-i f.. r1 ~" ~.1 r-1 .i. t..1 .C
t~ 0~

V7 a H H r1 fl. x ~ ~ U' H W G1~
W r-I

rei " j".. U7 ~1 .~'~ ~
~ ~ f' O

r- I ld ~, U) -ri ?~ rI ~-I
I r~ ~ 00 ' ' V, Ur a ~ Rf, x E-~ U' ~Q U
~-i~ . T ~

~ o ~ ~ ' Gl al A1 it ri ri r-1 r- U1 ~1 Ts n0 I

tn h~ a ~ tll 4-Z U' ~c4' H C7 ~C ~
,c~ r-a ' ~ ''~

~ , r~ ~ ~ ~ N ~ ~' ~I
~
M

~ ~ tLl fd QI r1 .C~" ?~ UJ 01 CO

ri eel ~ ~' > E-~ a x ~.e U
~ V1 r-~

x H
-I

~ p ~,,E ~ "~y r-I r1 r1 r-~1 N H
CO

~4 C7 e~C H t~ a ~C C7 ~ H ~ L7 ~ ~

~ , ~ a ~ ~ ~ N ~.1 ~ ~.1 ~ 00 O
~D

H ~ ~ .G ri ~-i ~r O) !~d .G S..i h CO a c~ ~ ~c w ~ c~ N a ~n w N ~
~

Q ~

~ ~ ~ rlf Q1 N UI ~ ~ to O
a "

~l Q1 ri ~ e-d .t:: '.~ r'1 UI CO ri .G
CO ~.

r ~ ~' ~~' ~ U x ~ w~, c~ a, ~

~ a ~, ~ ~, b H c~ H ~ ~ a tai ~ ~'' '1 ~

~ o .
~ ~ ~ O ~ y,.~ ~ t?. ~a ~ ~ O ~t ~ ri ~ s ~ ~ C cn .~ ~ ~n ~a a, co ~ ro -I c~ . . ~c N a ~ ~ ~
r a N w ~

c~ c~ ~ ~ _ ~

~, ~, 1~ ~ a~ ~ ~ m ~o ~o m ~., ~, ~., s~ ~ ~ ~ ~n ~-, r., m c~ co ~ ,~ ,~ ,~ ~ a c~ a ~
,~ ~

o o ~ ~ ~ co ~ s.~ a~ ~ ~ 0 ~o . ~ H ~ W H
~ ~

, ~ U L7 ~ C7 C7 E- 7 ~

~ N tff fa.e ~ U7 O N ~ t~ Q1 Ov ' ~y ~ r-1 r-i N rt ?, N -ri e--I .G N
t~ CO

a a H ~ ~~ U' a a x n ~ ~ o ~ ~ cn ~
~

o~ r~ o ~ r1 r-1 UJ .G' N ?v H ?h H
CO c0 p E~ C7 C~ ~ H ~ U C7 E1 C7 H e-~1 , ~ ~
~

o ~ ~ o o ~, ~ r, a. o~ o N

-I ~ ~ N ~ ~ ~ rt s~ rt1 t~ r-, rn r~ m ' '' H H r1 U' (.7 t~ ~ C9 W Rr J J r -I H

;~.; _- ,_~. _ .; ;, .. _. ; .~ : :-' ; .- ° , _., :::. -,.., , .
.°. .: ,,.-:
~.~,. .... , .. ., : . . . . , .,: . . ~ : . .. . . , . .. .. . . .. _ . ,~ .
. ., , W~ 93/20~93 P(.TIUS93/03275 _7 w 5~

N ~ N ~-1 N o S-~ ~ :y 6?e o 00 H N "~ ,-'~ ?r rtf H S-~ .C r-I
~ O

a c7 vN ~ H w H HN a H

~ ~ ~
~o ~o -~ ~ ~

c~ a w c~ aN x ~ cn ~ cnN a ~ o Q

u~ tr ~ ~-, w ~ o a. ~ i ~
~

' , r .
N ~, a ~ ~ ~ a w cane H N w cn aN
~

yr ro ro ~s a~ ro ~ ~ N a~ ~ ~
a~ o cI~ D C7 ,~ a cn c7 ~ ~ w C7 ,-a ' N

~ t1~ !~-1 ro ?i ~ ~ N
o ~
t0 tp a~ ~ .~ ~ ~ .~ ~ ra N a~ ~ N
a, o a ~

a ~ ~, ~~ ~ H ~ c~ ~N a ~ ~

c7 ~ ~ N H cn ~ E~ H a c7 a .-t N

a ~1 r4 ~ 'ir p 'Jy C ~-I ~ a ~.I
~J M dJ ZT

f.1 01 G7 td G7 w-t A) r-1 r-I N N f-t Ol O O
' "~ a ~ ~'N ~ ~ ~ ~ N 'V1 R~

~

~ '~ N ,t",.~ ~ ~ N ."'r 9r r-i t6 f.~
~' a .r! N r-I rd ~ O r-1 o-d ro H ~ H

~ a ,~ x ,~ ,~ c~ a N c~ c~ ~ ~ H N
o o !a N ro o i-~ ~ N ?s d, ?., ~ ' vD

La DD r-+ ?~ '.~ H ~1 ~ N ~r r-t CSl O

. a ~,~ H a ~ ~, ~nN N a H

~ ~ ~ ~ ~ N ~ ro ~ a~
~ s~
~ ~n N ~ o o o .~, ,~ ~ a: ,-., r., ~-., ~ o ~ H ~ a~ ~' x H ~ NN H ~ H

a o .a.7 p:, 4I PiS ~ ~ ~; ~-1 N tl~ ~d Q1 l~

~ ~, ~ ~ ~,~, a ~ ~ x HN H H

~ ~ u~ .~ ~ ~ N a~ ~ s~ ~
a~ ~ N
o c~ ~ ~~t p~ ~ .~ ~,, ~ a~ n~ ~
o o H~, ~c x ~ a x~ ~ H a ~n aN ~

o o a ~1 N d' ~ N ~ ~ N N ~ t3'~ Q? ~ '~ Q

'~p -1 r1 ar N r-i ?~ r~-W .1 .Li r-I r-!
f71 O ~-1 ~ ~.-~ 1-t a ~," C7 a N U' ~' G~ H C7 N
e-t ~ ~ '~

01 ?~ r1 r-i N .C r-I ~-1 r-O I
' C7 ,c~, f4 E-~ C7 U' ~ G~ ~'' ~ U N
e-! N

Q O

f ~ N ;.1 r-I ~ S-i f~ 3 "~ N
h tt1 , r-i .1 r-1 id ri ~ N ~ i-i .-.1 .,--!
,~ (35 O
N

x C7 ~ ~"'/ ~ ~cL,'a C7 x H
~"~ N

~ ~

t-~ ~ ~ L7~ N N
t0 ~

r-1 N r-1 r-1 .C. fv ?, S.-~ 9, ?i r-i ?~

H ;~ H c~ H~ a H ~ a aN H

V1V~ 93/20093 PC~'/US93/03275 ...., -~7-o ~ ~ ~ rd c~ ~ ~ ~a ~ oa ~

r1 ri N ri O r-~ r1 U7 ftS '',~t ~'., fll ~-1 ~-1 N

~ H ~ N ~ ~ ~ ~ P H H
N

(U (~ ~ ~ ~ UJ r-1 N ~
S1 .N
t'~ ttl e: ~.1 ~1 O ~r ''I t~ :Jy A1 f-1 c--I ~: UI
ri N

. w ~ cnN H a ~ a ~N H c~ H a a, n~ cn o ~a c .N s~ o ~ ~ rd N ~
a, ~

;~ ~ r-.~ ?i r-t O r'-I N QI .-1 N 4? H
.-i N

Oa R~ H U H tn C7 ~ tn C7 ~ a ~C
N ' N

-1 r ~ 1 ~ ~ O Sa 's"~ ~
S O
O

e . .1 - ri r1 O ,'y N U3 O N r--I S-I ~1 ~i .~; N
ftS r1 N

~r N ~Gi fn (n C~ W Lh H H .'h U a H
N N

- .i.~ O S~.1 C ftf ~-1 O U7 ~ ?s ~ O
Q1 ~ O
'd' N O

O ~, ~ ~ r1 47 'H '~ Qf r1 r1 S~-1 r-1 ~ N . M

v'~" W H ~ ~ t/) ai a a U' U" 63a L7 N N N

QI ~ :3 f~ f~ ~r ~ ~ f~ O ~
Il9 !"..
tn c'1 .r1 ri Pi O IlJ ~-1 x-1 r1 N SO .C ~d e-d N

a H x N a ~C H H C7 a ~C L1~ W
N

a ~r ~ r~ o ta~
c~

C' tn C Ul O .C P-I ,l.,"Ql r-~i fIf Ul fd r1 N

, meC . ~'. ~1 H ~ W ~ N U' '~' ~C
, f_-n N , E-E

~ ~

u~ a~ ~ ~ ~ cn ~r cn a ~ ~

s.r ~ r-a ~ m N a., ~ ~ a~ ~a ~ N

c~ x s~ v HN ~~ ~ ~ a cnN a a H

~ ~n cn ~, ~d o s~ ~--~ ~ s~ ~
~a N o ,-~
o ~ o ~ a n ~N ~ o HN a x ~ ~ ~ c c n n f"0 ~ ~ ~ ~ .1-~ CT ~ ~r ?a t0 C ~
M r-t ' 01 ri Q7 N ''.fir(U 'ji ~ r1 N ?t ~ ri r1 N N ' tn H a ~', a ~ ~ C7 C7 X13 a ~ U
N N N

~w ~ ~ '~ ~-I G" >r ~ ~, O ,~., e--I () fV't ~

Ul r-1 ~ r-I ~ f11 U7 r1 O r-I r-i ri (d N

~ V, ~', a ~ ~ ~ ' C7 H C.7 ~ ~
N ~
N

tf! ~ ~

~ ~ ~ H ~., r-I S~ .C O ~illJftS '''1 H d N

t cn H ca t~ H c9 H H N a ~ ~ ~
N , , ~1 S.1 ~ ~ O ~ ~ i.1 o ~ O O
$f~ $~ O
~ lD

S-a N 'J~t N t17 .~' N r1 .~: JL; e-1 w-1 N

~ ~ H ~' ,~'" ~ a L9 H W C'J L~ H
N N

~ G~ ~ ~ 'fit ~ U7 UI ra :3 Ql 1.! ~5~
~d r-1 S~d t'~
61 "

r-I 'fir r1 ~ tfs ri N .O ~.1 r1 .~

HN ~ ~ ~ c~ HN ~ a a ca a~N H

o o o r-1 ttf ~ ~ S-1 ~ O ~ (f7 O N ~
~ r1 ~

[ c ri (U O DI Ql ~-I r-0 ~ .C: .C O
" t~ N N
r-9 . ~N ~ a a cn aN a~ ~ a w a~N a , H

~ ~' ~..1 O 3a b~ .~. ~..1 r-1 '.~ ~-1 ~-1 cY N

r-i . r1 r.. ~1 ~ ~.1 U7 ,.C.' (l7 N (U
Sn v-i N

,c~ H N . Q., cn ~ ~ H ~ a tn H
H N

~Y~ 93!20093 P~'T/1JS93/03275 ;, vD N CO d' Q

-~ ~.-1N M f"7 N

N
H U U H

H ~ C C
.7 7 H ~

U U U

~

U

.- ~ V

H H

~.

J U U H

H E- ~ c.
~ .~

~w H
H ~ ~

U

~ ~

~ H U H H

..

~ m H ~ H U ~ H
~

O H ~ ~ H .C7 Z ~ H C7 H C

U H H

N ~

.. Ot C9 E~ C7 C7 .. U a't'e ~ tJ~ ~ U H
fT

r-i u1 H N -I ~ O U U C7 U C9 ~

.. H ttJ U ~ ~ U U U
w! ~

~ r.~ H ~C7 ~
' ~~

Z o. U O H U H C7 H U-N
-i~

~ V " ~ ~ y~ H H H H ~ ~ U
' N U Q W a ..-1 'z, O H L9 C7 C7 C~J J U
V1 r-i C

~ H E, ~ ~ C7 O .. p,, E., ~ ~"~c9 C7 C7 t~1 ~-.I p, ~, U N ~-i ~ W H U ~ H
N W

,.-ir~Or ~U~ .. O U t~ U H H U H H

~ W ~ ~ ~ ~ W ~ GY U
N ~ ,~ .. C ~ W7 ~ tI~ H U H ~ U
W L7 a tn W U U ~C C9 H

U p4 x x G1 ~ ~ .. p D H U ~ H
O H U W UJ
U H Z a Z

a O O W ~ O H u~ ,~ H ~ C7 ~ H
w ~ ca c~ ~ U

6z, W :~ ~.a W H z a c~
W

r! U W >e a W W ~ p4 U L7 H H ~ H C7 H O

c~ ~ ~ ~-7 H a ~ tn Z O z ~C U H H C9 en H

O W U H ~ y-..i W . U H H H H
U

H ~ ~. ~-. W O H U' .-.... a ~ ~ ~ ~ ~ v ~ H ot~r~Ua ,a w H H~

W v v w. p .'Yt.7..~ ~'' W
~ H

L7 ~ en ~ ~ FC O U 7 H U ~C H
C

H . ~ ~C E-aH
- H

~ ~ ~ ~ .~ .~ j ~ .-I H U H H C7 C7 .,a .,.~.~ > ?C U U c.~H C~ H

H ... .,.~.r ...
~ U

.... E., [-~ C r~ H

N
U U U

~ H

l~d~ 93/20093 PCTI US93103275 N co er o vo N co ~r o o N w ~r a yp op ~ ('. pp 01 01 O O r-1 N N
r-1 ri e-W 1 ri H U U ~ H t9 ~ ~ U U U '~ U
H H C7 H ~ U ~ ~ ~ H ~C U
H U ~ ~ C9 ~ C7 U ~ C7 ~ U
U H H ~ L7 ~ ~ t7 U c9 U H H
H U ~ ~ H U H U U C7 KC H H H t7 ~ t,~ H ~ ~ U H H U U ~ U H
E-~ U U C.~ H H U ~ U U U U H H
H ~ c7 C7 c7 H ~ U ~ "H ~ C7 H C7 U U ,c~ t' ~ U U U H H KC U
C7 U H C9 U H t7 ~ c? c7 H U C7 U H U U ~ c7 H H H H C7 H U
H U U H U H ~ H ~ ~ U ~ ~ H
C7 H H U H C7 ~ U U U H C7 U C9 U ~ EH-, ~ ,~ ~ H H U H ~ U U
~., H E-~ H H t9 C7 C7 H ~ ~ H ~ ~ ~
H H ~ V ~ U U H ~ C9 ~ ~ C7 U
U'~HEU-~UU~CU.7c.'~h~NU~H
C9 ~ ,~ ~C C7 U H H H
U U ~ ~ H C7 t,7 C7 C7 U H
~ H H U U C7 ~ ~ C9 C7 H C9 c9 L7 C7 H ~ t7 ~ U
~ C~3 E ~ U ~ L7 U C9 H C7 U C7 H U H H C7 U U H U H U H ~
U H ~ H H C9 ~ C7 C7 U H ~ U U
H ~ U U U H f9 ~C ~ H U C9 U
(,~ U H U C7 H t,~ C7 C7 C9 C7 C7 C7 C? U
,~ H ' H r~ C7 C7 H U ,~ H C9 U ~ U
~HL7Hc~c9HUC7U~~c7C7~' ~~UO°~
H ~C H c7 ,~ H c9 H L7 C7 U AC ~ H ~o M ~ ~
H U H C7 H L7 H L7 U U U U U U H -~ 'L~ °~
H ~ U ~ H U ~ H r~ U H C7 C7 U a~ d tl~ ~ ~-i U) U U H H H U H U E, U C9 ~ U t9 C7 Z H b U
,a;HUHUH~CHU~C7 UDC fx ttf,~~
C7 H L7 H C7 C7 H H U C7 c9 C7 H L9 D W O Cn H ~ U H H C7 C7 U U H H U U ~ H U o G W
C7 H C7 t9 H C7 H C7 U H ~C
E.,. ,~ H H U H C9 C7 U U C7 ~ U U U
~ H ~., C7 E~ U L7 ~ ~ C7 H H U H H t~ ~ ~~ ~ W
U,y~Ur ~,c~,~,,~UC?~CL7HUUH '~,.~'Ll U H C9 H L7 U U C9 L7 U ' C9 AC U hG U H
H ~ H U ~ H L7 H ~ L7 H H C9 H U O C7 W
U ~ ~ U U H H U H U ~C U H ~ W W ~ ~
oQ; U U ('~ ~C L7 U ~ N t9 H L7 ~ U L9 U W ~ H
C9 U U U U ~ ~ H H U U H U ~ ~ ~ z a H ~
U U U U H ~ ~C U U H U f? W
E-~ o~ s~' ~ U H C7 ~ L7 U H C~ H 6-~ ,.~ .-. ~..
,cy' CJ H H H U ~2 C9 H U ~ ~ ~C 'C H Ot ~ CC~ U
Wvwv U H ~C C2 U U U ~C ~ ~ H U C7 C7 U
U H H c~ H H C7 c7 U C7 C7 H H
,~ E-, U E., U ~ ~C H U ~C H U ~ ~ E' p ~, ,rs, ~ U H U ~ U ~ C7 U C7 h. -.-I
~-/ H U ~ H ~ H t7 C~ ~ H C~ L7 r.C H v ,c~ ~ ,~ H U C7 H H ~ ~ ~'C U H
U U H ~ C7 U H U H U H U L9 U U
H U C7 H ~ H ~C ~ H U U H ~ C7 H ~
H C~ ~ C7 H C7 U U E-a ~ U a; H C~ N
E., U ~ U .~ U C~ ~ c7 c9 C~ U C7 H ''' WO 93/20093 PC'rl~JS93103275 A~~:'~, s ..
~ ~ ~ ~ ~ ~

r- U f O r- Ul i l l t ~

C9 LL ~ C7 ~

~

~ Z7 t!~ f~ ~ a lT C7 e, ~ f1 ~ ~ :9 ffS ~
O

~ H a ~

~ a ~
~n ~ ~ a cn a. c~ a .~

~ ~ O ~ a .~ ~ ~ ~ c E
,. n -H H
~

~ ,~ c C9 .7 ~ ~ ~ ~

~o ~ ~ o w D t/~ U ~ ~ H C9 cf3 ra a~

O ~ ~ ~ ~ ~ ~ b a ~ H r r-N a c~ c ~

~. 9 -t r-, a x H ~ cn t7 ~

~. ~ .. ~. ~.

~ ~ ~ 'V ~ ~ N ~ O O O

~ O d-~a-t e--I r1 ?~ :err S.a r~i ~-a N
, o . O Ea C7 a H ~ C9 ~ t!) t~

~ t~ o z w ~ H ~ ~ ~, ~ m as o z (~ p; O tv .~ H ?~ tn .. ~n z ~ ~~ ~ a~, a ~ c~ a ~~o a~

~ ~ a w ~ ~ ~ a~ ~ ro ,.

o ~. ~ ~ a~ o H o b a~ yr ~ .-~ cd rd v w H cn o o z a H ~ aN ~ H ~ x w w ~, z H a~ w o a w w z ~ x U s~ ra a ~ a x cn w z o z ~ r, u~ c~ a~ o~ a~
~n U H 1 ~ p..i w E, ,~ ,a~ ~ cn a cn M

W ~ ~ ? ~ ~ ~ N

1 . ~ o U C

~ x ,~ G~.~O tl~ a H H ~ ~ C7 a ~

.-. .-.
~ ? N

:~ ..-I~ a ~ -~-t m- ~ ~ r tI~ c11 x c~ ~ ~ a cn ~

.....~, .... ~. ,., .. . . , ,: . .. . .:_ ..-. , ,. , rt _ .:- : ;...
::.<,,~. ... .:: ..s-.:.;..~. . ......., .:,.... .~-.. . :- ,.: _ :..,. , x,!, .--~ . .. : ~ .:

~V~ 93120093 PC1'/US93/032°75 r..., , b-1 1~ fl1 L/3 ~'1 O '' O

r1 .~ td e-1 RlI
~ a 0. d' E' ~ x a c . t7 E- x c7 -~ ~ n N

tr ~ ~ n~ ~ 0 0 0 ~ ~s a. ~n ~, ~, cn ar ~ a~ ~-, sa a~ mn r~ cn r en x cn w w a cn x ~ a ~ N

it s~
a -/ ~ -r'i KS U7 r1 r-I (IS ~ f~ Q7 .~, Q~ h s~' Q: x: x Q; H C.~ > U~ x tl~
ri ~ N

r-I N r-I -rl f0 N .f"..~7 '-1 N (U r-1 O CO a (7<-i U x, x a HN a H a x c~N

~ ~T ~_J f'd Ul O S-I fC O to r~ S-1 O
' O ' O

.. ~..I . i(S ~ ~-d ~ r-! ~-1 (D ft3 GJ O
r-1 d' 4I N

~ ~~ a x a w cnN a w x >

s u~ ~ ~ ~ i ~
~;

. s .~

~ x a ~ x c~ > x a, N

c~ ~. o cn ~ ~ ~ a ~, ro ~ o o o o x ~ ~ ~ ~s a~
~

~ ~ ~ a a H H a t~ x cn w ~ N

o ~ ~n m rr ~ ro ro ~, >., ~

m s~ ~ ,~~ ~ ~ ~, ~ ~, ~, u~ as ~c c~ ca a ~ ~ ~ x >
~ N

~, o ~ ~ o rd ~ ~ ~ ~, res ~ o cn o N N O ~ ~-I (if Q7 r-4 O r-1 (I1 U~
O CO

tn P-f a a W x a C7 !n CJ x ~ N
N

~ ~ ~1 ~ ~ ~ 1~.1 ~ N is :~ td t!1 ~ t(1 ri (d 41 .~; ~-1 r--I ttS r~ r-1 O~
H

,,~ Er ~ C~ x a E; ~eC H x c~ ~ N
rt N

p, td O fa s~ ~ v ar ~ ?~ ~, ~ O la (n: w-i f-I t~ ~l1 ri .~i ,Li f~ Pi $.1 lf~ C1 .~ w x ~ ~ c~ ~ x ~ ~
~, ~.d fa ~-i f~ ~ tll f~S
Il'1 lIl r-i .~ rtf ~ r-t tip -~i td N
t0 ~r ~, x ~n ~ ~ x x a e~
~ N

N (~? nj ~ (t1 ~-1 S-~ r-t S-~ N ~-i U7 O O

.~ ~r ~, o~ ~s ~ ~ ~ ~ ~ ~ a N

~ x ~ a ~ c x o ~n rt ~ ~ ro ~a o ~ ~r oa ~ ~n ~
~

m ~a as a~ ,~ o ~ ' ' W r-1 ,'~C Ll~ > x U7 a U' C4 ~L (~ V
r1 N

b it o t~ rcs s~ ~ o ~ ~ ~ a~ o o o ~, t~ ~ c~ a~ .c ~ m ,~ ~ .~ ~
~, ~c ~c ~, ~ x cn H w ~ w c~ w N
~ N

~ ~ ra .s~ f~ tm.n m ~ a3 (U
try ,~.r r1 r1 fI~ ~ U! r'1 ~y H ~ '-1 d' N

~ C~ x ~ ,c~ :~ a U' x H
N

~ ..... . . :.:: , .. :. _ ,, ;, . : : :. ,.. . .. .... ;:..
r~,f ....:.',~., ",~:,. ,".~~.v. . .o: , ' ~.~~.,. . ~.,. ;.:'..~ ., . .:.:., . , . ,.,.; ,.. ,,.~.;'. : :..:', . .:..... :,. :. .; .,~ . ~:, "..~.,.a'..
~;.~; .:.:..u. ' .~:..' . '.::a . .. ,:.. "... ..... .,..~... . .., ..,... , .., ~~.. .: y ........ . ..-:.
... ~ .:. ' ~::., . , .. ... , :.:, ,~ :.;_, .,. :.,. , ..,...,,.,~. :.. :.,:
a. ,.. .. .:...,....... ,. ~.. ::~:!., ,.,... . ....,.... . . ... .. ~;:. , ., ,......,.. :..... ,..,. ....... .:,:.-, ..,..;.:. .. ,..:.,.... "" ~s.~ ..
~..~n.,. .....:. , . -,......,..:....
?' .:...,. ,.......,, ...:......, ..,:"."... ..,..,.....:. ....,., . ...:...~.
.... ........, ..,........, ,., : : . .:..:.." "......:....,. .....::..., ~.:......,. " ,,.:.. , :.
~.~:.m...,:.,. ......... .,.,:,.; "."......, ....,...... .,.... .,. .......
.:,.. ,., ,,..:.. , ..:...., ,....:.~ ::. "..;....... ,: ,.,..< ... ~ . ...
.,.,_..,: ,..,. _.,.
...;w'..:., ~ . ;.~,..,t , , ",.....: .!.°:.-.~'.~ ~...~_ .~..., .. ,., ,....,.,.. . :..,...,.. ~.....,;...: . .,..,,;..~. .,...... ,.n _..~...... ...
.. . . ,.
W~ 93120093 PC'~/LJS931~33275 sF~t?, _02_ ~'~, °?' .
roa ro ~ ro ro roo ro N ~r rr roo r-o N r-.i ~ ro rti ro ro >, ~ i~ it o c~

,~ ~ ~ . x x x x a cn ~c x N
~ ~r ~r N rain ~ ro ro ro ro~ ~, ~ ~, ro c~

-~ N ro ro ro ro -~ .~ ~ ro N
c~ ~r-W or a ~C ~ x x x x c~ H ~ x c~ ~r Via N ro it ro ro ~ ~ ~ ro , O o N . ~; ro ro ro ro n~ o ~, ro N ~ ~, ,~ ~ a x x x x a a c~ x N
M ~- ~

a~ ~ ~ ro ro n~ ro o ~ ~r, ~

~ N N ro ro ro ro ~.1 4I r1 r-1 vD "C' , ~ ~ x ~ x x x a~ a ~ c~ ~ a " ' ~, N a~ ~ ro roo ro ro ~ ~ ~o N o r, as ro ca ro ro r, ~ . a~ ~ .c co ~

a H ~ x x x x c~ c~ cn a w M ~

N n~ ro ro ro ro ~n o ~ ~ s~
~n ~n ro .-.i .P.a C co ro ro ro ?. s~ ~ N
a. c~

~M ~ . x x xr~ x a w ~n ~~ H
~

~ vo ~, ~ ro ro roo a~ s~ ~ ~ ~o 07 v-~ C. N ro 10 ro r-I (v .-I
M P-i a H . ~ x x x H V3 C7 a ~-l CI ~ ~ d' ~' ~ N a0 9.a ro ro ro ~ ~J ~ ~ , u7 ~ =a O? 'fir r1 N PLf ro ro S.1 ~.d r-1 r-I r-1 'd' N

N a ~ N x x x H w M ~r -+ r~ ro ro ro ~ o N ~ s~
o r--~ ~., ~ ro ro ro ro ~ ~ ~r ~, ~ a7 ~

H ~ cn ~ cn x x x x ~ ~ a G7 N

~ ~ ro N ~ ed ro ro ~ ~
,~ ~n N N ~ -~ ro ro ro ro s~ ro ~ N
c~ ~

~ ,~ ~ x xM x x x r,o s~ ~. ~ ro ~o ro ro ~ ~ ~o 0 ro~ ~ ~ N ro r~c~ ro ro ~ o ~~ N

E~ ~ ~C x x x x c~ cn ~ w r, ~r N N ~ ~ ro ro ro ro S~ N r..i ~ ~c1 ~ ~ N ~ ro ro ro ro N ?r ro Q1 N O CD

a a~ ~ a x x x~ x cn a ~ a~

a~ ~ ~ a. ro ro ro ro ~ ~, ro .s.~
o o o ~~r ~ ro ro ro roN o ~, a ~ e~ x x x x cn ca ~ -~

~ ~ ~ ~~ ro ro ro ro a~ ~ ~ n~ b ro ro ro o ~ v ro ~

a H H cn x x x x a ~r cn r ~

~ ~ ~ ro ro ca ro ro o ~ tr o o ro ro u~ ~ ror ro ro ro a ~ x x x x a, c~ ~c c~
~, ~

t~ u, rn ~ ~ ro ro ro ro ~ s~ a~ N
~ ~

N o ~ ~ .~ ro ro ro ro N a~
co a E-r x x x x ~ ~ H a M ~

W~ 93/20093 ~CT/~J~93I03~75 a3 ~~ ~~~~i~i ro ro ro ~o rn ro ro ro ~ a ''~ C ~ X ~~r f~ ro x ~ 7 ~ ~C x a a c 7 C
-i n C

ro~ ~ ~ b ~ ~~ ro ro N i c in x ~ ~~ x x ~n. ~ ~ W 7 C

N O ~-i tT ~-, i.~ ~ ~ ro O ~ ro ro O O ro ~ ~., ro y~ ~., v r, s~ ro ~ v ro x o, ~
x ~

a ~ > ~ c7 cn t7 ~~ x w en nn N ~ s~ s~ ~ o ro ~ ~ ro ro ~n rn ~n 5. aw a~ N cn f~ ~ ro v .~ ro ro co N o x x a a~ a ~ ~ ~ w~ x a H

~y :3 v o A) ~ ~ ~ ~ ro ,-i ro ~ ~ !f3 x N x x~

c~ , ~ ~ ~ ~,,n o ro o ro v u~ ~ ro ro ro ~ ~ N r, v ~ ~ rd ,-s m ro x m r~ ~ x H ~

ca ~ ~ ca a ~ r~ x ~n cn ~ ~o ~a ~ ro ~ ~o ro ro .~ v v ~ ~ ~ Wn ro ro r.

cn m C7 x t7 a x ?C
~n ~

N ~ , ~ ~ rn a~ r~ ro N ~-, ro ro ~ ~n ~, .~o v .C ro ~ ~co ~ ro ?, ro ro~
o x x~n a ~ ~ ~c ~~, H x a ~ x~

tn N to ~ ~ ro ro o o r: fLS r1 v v -ri .s; N fu ro ro ro CO
N O x x ~

. x c~ cn en x w rr~ a ~
~n ~

O'' ~ N ~ b ~ ro ~

r ~ a x a ~ ~ ~ ,~ ,~ ~ x c~ x ,~

ro ~o ~ ~ ~,, N roo tr ro ro N ~ ~ ~~ ~ ~ ~ ~ roe, ~ ro ro ~ ~c ~ c~ ~ H c~ a x ~ ~c x x ~

~ ~ ~ ~ ~ cn o ~ ro ,~

a a x x , cn x ~

~ ro tr ro ~ ro ro ~ ~ ~ o o o ro .~ ~ ~a ro ~ ro ro m vo x cn ~ H ~ ~ x a~ x ~n ~

, ~, ~ ~ o ~ ~ ~ ro m ro ro ~n nn ~, r . ro ~ ro ro~

~ a ~ ~ ~ c~~, ~ x a x x~o n t~, ~ C o cn ro ro ~ o ro ~ ~ ro o ro N ,~, ,-~ ..~ ,-~ ~ v s~ ro ~ v x r, ,~ cn ~ ~ ~ ,n x ~ ~ a w x H
.o ro ~ O v ~ ~' O ~
in . S ro ro ~ ro ,.i i-1 ~.1 t.' .~ N
~P

r ~ a, , x ~ w ~ x ~ c~ H x ~ a, ~n !WO 93/20093 PC'6'/US93/03275 a''~''~, _84_ .., ro ro ro ~ ~ ro ro tT N ?a ro ~'' O O ~

. ro ~o U

x x x a C? ~C ~ ~ C7 x C9 00 c~ ~

ro ~ ~

~ ro ro ~ ~ ro a~ ~ ro a~
~ ~ cn ~ x a x x x H a ~ v~ ~
P ~o ,~ ~ ro ro o ' ~ ~ ro ~d :~i ~ a7 .7v ro ,h ro Q1 lfl M a x x ~ w ~-, cn cn a > H x ~. ~ oo ro c0 tU ~a ~ ~ N ~ O ~ N it1 ro N

c~ ro ro v v ~ ~, cu ~ N ~ ~ ca x x x c~ aP a a w ~ aoo x a ro O ro ro s~ ~ ~

o a cn C9 ~ cn ~

ro ~s ro ~ o ~ ro a~ s~
~n ~n ro ro ro ~ s~ ~ ~ ~ .~ a~
~, c~

x x x c~ w a ~ a, H a ~a a~ 00 P P

ro ro roo ro ~ ~ ~ ~

, x x ~ x a ~ a x~ a w a~ H
~ ~

ro ro ~ ~ ~ ~
~

y -I ~1 r1 ro N

x x x x t!a t~ U' ~C C7 H ~ x t~ OD

ro ro ~ ro ~ O ~ ~ N O ~ O ~ ro r-H N ro ~ ~ ~ ~ d' -x ,~ s ..1 f- C~ AC x ~ -~ ~ 7 ~

ro ~n ro ~a ro ~ u~ N ~ ~ ~ ,~ ro ~n ~, ro~ ro ro a x~ x a ~ ~ a~ c~ a c~ >co x c ro~ ~ ro ~ ~ ~~ ~ ~ ~ ~ ~a P cn a ~ a x ~o ro ~ ro~ ~ ~ ~ ~ ro~ ~ ~ ~ ro ro ro ro ro ~ ~--I n7 .-9 N -3 N ro ra O

x x x x ~ L7 cn a: ~ H ~C x r- co ~ ro ro ro ~ ~y ro ~ ~ ~ ~ ro O O

ro ro ro ro : v r-I r1 r-1 r-i N
d' N

x x x x a ~ ~c c~ c~ ~ a x P ~o ro r~ ro b rou, ~ ~ ~ ~ ~~ ~ ro r, ,-~ ~ ~ ~, ~ M N ro u~

x x x x ~ P C7 a U1 ~ C7 ~ x ro ~., ro o ~ N N ~ ro a o ~ ~ ro ro it ~ ~ s~ ~ --~ -~ ro ro r ~

x ~o x x x c~ ~ a, a x x ~ x ~ oo ro ro ro ro ~ .N ar ~. ,-, ro o ro ~n ~, ~

~a ~ ~ ro ~

x X x C.7 ~ C7 C7 ~ ~ W x o0 ~ t~

r ,. ,:- . . ~:. .,.._ r- - . ..,. _. . ..;: : _ .;; : . r: _ ... ,, v;; .:-:... , '~".::..; :':'.,. . .. .: ":,:'.. , . :.~ . :. ' .. - .: . , ,.- -v , , , , ;..,, .'-;, , '. . ~, ~.", . . . . .,v-:: ~ ,:: , . .;,,.. . . .

'bV~ 93/20093 Pi.'T/US93/03275 ~~ ~~~~~3 ~ ~o ~ >, ~ ~ ~o ~ ?.
a r O~ tWo ~--~ r-i N O
a H H a ca c~ cn a ~c c~ a a~ ~

cC tt~ ~-1 fa ~d O O N
tt~ ~ r..d ~,./ r-1 N
a, a C~ ~ o ~C o n.~, c9 ~ -~

~o N ca o ~ ~ o 0 ~, -~ r~ !~ ~ .C ~, ,--~ N ~ c~ o ~ x ~ A., L7 a~ 4. c7 ~ ~C a r' d pC H ov"'lf1 or ~ ~ N O ~ ,.~ ~ N
td ~r, H ?~ S.~ ~ o rt5 ~-a .C a x ~ N c~ a w ~ N ~o ~ w O N ZT o ~'1 a O r-1 Q
a ~~ O ~ ~~ o ~ -1 ~ ~ ~ r-1 ~..a~ c7 ~~ rti o ~ ~~ s~ ~ is , in ~d ~-a ~ id M rt3 C ~ 0-1 r-i y, N ~ r-~ r-t r-1 C9 lf1 N o H C2 ,aC
o -r1 .L.~.'~'~ N .Lr'' r-~4 r-I ~ ?a x oo cn x H a p c~ c9 H ~ o H u~
a ~ ~
o~ ~

c~J (1l L~ fU ~ ~d d7 i1~ QI o N f.i vD
~ 1I1 s~ .~ n~ cu !fS N .~ i~ ~ a~ o x ~ ~ w a cn ,-i ~ w w a u~m ~ co 0 ~ ao~ s~ ~a ~r v H~ o a~ ~ m o ~ o ~ ~ H o ~ ~ ~ ~ ?a L~ w ~ N N
~ ~ ~ I~I1 ~ N ~ O r-1 -1 ..~
~ ~ ~ r1 Z7 ~C ?r ?, r-i c.9 ~cC ~
Q Q r'1 N r-i O ~
V~ as tJ ~C U x fT ~

td N Q ''~,ao ~ ~.1 ~.1 I3~ ~d O 1..1 r-i o ~1 :'~ :~s f-1 M b-1 . 'i.' x ~ ~ ~ ~8 ~ H H ~ O p.~ H
If? O ~
a, cn ~ ~

"~' ~~'q ~a ~ Q ~1 -N r-1 Q
',3y a0 r1 N r-t m-! lf'1 N P-1 .G fU ~d' .G
r7 ~ tJ a C'? C7 r-~1 ~C H H f~
to O
U' ~

,~ ~ ~~ ?~ N .N ~ ~ ~ r1 Q O
O O N r-i r-1 O N
a., Iy cn ~ L7 x Q3 ~ ~C C7 ~.7 cn ~--i O c0 a ~ ~
o~

'N ~d ~ N rQ-1 ~ 7r ~.~r ?~ ar r-~i ~ a c~ ~ a O~1 a H c~ c~
~ a ~C N ~ i~ s~ o i~ ~ t~
...~ o o ~, ~-1 N v, ~ N N N
~ t.7 ri a ~ ~ say ~ ~~ ~ AC
,-~ f"1 N r-1 H ~o~. ~C O
L7~a a ~, ~ rti 1.-~ ~ ~ ~ N t~ ~ r~
~..~ u~ v ~ ~ .~ N
H ~ ~ ~ a C9 t9 t~ ~a c.~
~r o H H
a~ ~

VlrO 93/20093 PCT/US93/03275 ~,R.,,, _8~,_ 6~' N

N

a ,~

~, ~

a ~c H

~~

a ar ~

~o c~ ~

a +~ ~

a ~~

N ~

r-1 r-i x Ht

Claims (19)

We claim:

1. A purified hybrid factor VIII molecule comprising porcine and human amino acid sequences, wherein the molecule has procoagulant activity in an in vitro coagulation assay and wherein the molecule is selected from the group consisting of a molecule consisting essentially of a human factor VIII in which the porcine A2 domain is substituted for the homologous human factor VIII A2 domain;
a molecule consisting essentially of a porcine factor VIII in which the human A2 domain is substituted for the homologous porcine factor VIII A2 domain;
a molecule consisting essentially of a human light chain factor VIII subunit and a porcine heavy chain factor VIII subunit; and a molecule consisting essentially of a porcine light chain factor VIII subunit and a human heavy chain factor VIII subunit.

2. The molecule of claim 1, wherein the molecule has a specific activity greater than 20,000 U/A280 protein in aqueous solution when human plasma is used as the standard in a one-stage coagulation assay.

3. The molecule of claim 1, wherein the molecule consists essentially of a human factor VIII in which the porcine A2 domain is substituted for the homologous human factor VIII A2 domain.

4. The molecule of claim 1, wherein the molecule consists essentially of a porcine factor VIII in which the human A2 domain is substituted for the homologous porcine factor VIII A2 domain.

5. The molecule of claim 1, wherein the molecule consists essentially of a human light chain factor VIII subunit and a porcine heavy chain factor VIII subunit.

6. The molecule of claim 1, wherein the molecule consists essentially of a porcine light chain factor VIII subunit and a human heavy chain factor VIII subunit.

7. The molecule of claim 1, wherein the hybrid human/porcine factor VIII molecule is formed by substituting the porcine A2 domain in human factor VIII.

8. A pharmaceutical composition comprising the hybrid human/porcine factor VIII according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

9. The composition of claim 8, wherein the carrier is selected from the group consisting of stabilizing agents and delivery vehicles.

10. The composition of claim 9, wherein the stabilizing agents are selected from the group consisting of proteins and polysaccharides.

11. The composition of claim 8, further comprising clotting factors selected from the group consisting of von Willebrand factor, vitamin K
dependent clotting factors, and coagulating tissue factor.

12. The composition of claim 9, wherein the delivery vehicles are liposomes.

13. A method of preparing purified hybrid human/porcine factor VIII
comprising combining primary amino acid sequence derived from porcine factor VIII with primary amino acid sequence derived from human factor VIII to form a hybrid factor VIII molecule having procoagulant activity in an in vitro coagulation assay, wherein the molecule is a molecule consisting essentially of a porcine light chain factor VIII subunit and a human heavy chain factor VIII subunit.

14. The method of claim 13, wherein the hybrid human/porcine factor VIII molecule is formed by isolating and purifying heavy and light chain subunits of human factor VIII and porcine factor VIII, then mixing the human and porcine subunits to form the hybrid human/porcine factor VIII.

15. The method of claim 14, wherein the human and porcine factor VIII subunits are isolated from human and porcine plasma.

16. An isolated DNA encoding porcine factor VIII A2 domain having the amino acid sequence as set forth in SEQ.ID.NO.:2.

17. An isolated DNA according to claim 16 having the nucleotide sequence set forth in SEQ.ID.NO.:1.

18. A DNA encoding a hybrid factor VIII-comprising amino acid sequence of human factor VIII in which porcine A2 domain according to claim 16 is substituted for the homologous human factor VIII A2 domain.

19. A method of making a hybrid human/porcine factor VIII
comprising expressing the DNA of claim 18 in a suitable host cell and purifying the hybrid factor VIII from a lysate or cell supernatant of said host cell.